Pharmaceuticals

49 pages, 1247 KiB

Open AccessReview

Psychotropic Drugs for the Management of Chronic Pain and Itch

by Daria A. Belinskaia, Mariia A. Belinskaia, Oleg I. Barygin, Nina P. Vanchakova and Natalia N. Shestakova

Pharmaceuticals 2019, 12(2), 99; https://doi.org/10.3390/ph12020099 - 24 Jun 2019

Cited by 29 | Viewed by 9425

Abstract

Clinical observations have shown that patients with chronic neuropathic pain or itch exhibit symptoms of increased anxiety, depression and cognitive impairment. Such patients need corrective therapy with antidepressants, antipsychotics or anticonvulsants. It is known that some psychotropic drugs are also effective for the [...] Read more.

Clinical observations have shown that patients with chronic neuropathic pain or itch exhibit symptoms of increased anxiety, depression and cognitive impairment. Such patients need corrective therapy with antidepressants, antipsychotics or anticonvulsants. It is known that some psychotropic drugs are also effective for the treatment of neuropathic pain and pruritus syndromes due to interaction with the secondary molecular targets. Our own clinical studies have identified antipruritic and/or analgesic efficacy of the following compounds: tianeptine (atypical tricyclic antidepressant), citalopram (selective serotonin reuptake inhibitor), mianserin (tetracyclic antidepressant), carbamazepine (anticonvulsant), trazodone (serotonin antagonist and reuptake inhibitor), and chlorprothixene (antipsychotic). Venlafaxine (serotonin-norepinephrine reuptake inhibitor) is known to have an analgesic effect too. The mechanism of such effect of these drugs is not fully understood. Herein we review and correlate the literature data on analgesic/antipruritic activity with pharmacological profile of these compounds. Full article

(This article belongs to the Special Issue The Story of Successful Drugs and Recent FDA-Approved Molecules)

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Graphical abstract

18 pages, 1278 KiB

Open AccessArticle

Design and Synthesis of CNS-targeted Flavones and Analogues with Neuroprotective Potential Against H₂O₂- and Aβ_1-42-Induced Toxicity in SH-SY5Y Human Neuroblastoma Cells

by Ana M. de Matos, Alice Martins, Teresa Man, David Evans, Magnus Walter, Maria Conceição Oliveira, Óscar López, José G. Fernandez-Bolaños, Philipp Dätwyler, Beat Ernst, M. Paula Macedo, Marialessandra Contino, Nicola A. Colabufo and Amélia P. Rauter

Pharmaceuticals 2019, 12(2), 98; https://doi.org/10.3390/ph12020098 - 21 Jun 2019

Cited by 11 | Viewed by 5268

Abstract

With the lack of available drugs able to prevent the progression of Alzheimer’s disease (AD), the discovery of new neuroprotective treatments able to rescue neurons from cell injury is presently a matter of extreme importance and urgency. Here, we were inspired by the [...] Read more.

With the lack of available drugs able to prevent the progression of Alzheimer’s disease (AD), the discovery of new neuroprotective treatments able to rescue neurons from cell injury is presently a matter of extreme importance and urgency. Here, we were inspired by the widely reported potential of natural flavonoids to build a library of novel flavones, chromen-4-ones and their C-glucosyl derivatives, and to explore their ability as neuroprotective agents with suitable pharmacokinetic profiles. All compounds were firstly evaluated in a parallel artificial membrane permeability assay (PAMPA) to assess their effective permeability across biological membranes, namely the blood-brain barrier (BBB). With this test, we aimed not only at assessing if our candidates would be well-distributed, but also at rationalizing the influence of the sugar moiety on the physicochemical properties. To complement our analysis, logD_7.4 was determined. From all screened compounds, the p-morpholinyl flavones stood out for their ability to fully rescue SH-SY5Y human neuroblastoma cells against both H₂O₂- and Aβ_1-42-induced cell death. Cholinesterase inhibition was also evaluated, and modest inhibitory activities were found. This work highlights the potential of C-glucosylflavones as neuroprotective agents, and presents the p-morpholinyl C-glucosylflavone 37, which did not show any cytotoxicity towards HepG2 and Caco-2 cells at 100 μM, as a new lead structure for further development against AD. Full article

(This article belongs to the Special Issue Carbohydrates 2018)

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Figure 1
Chemical structure of chrysin (1) and 8-β-d-glucosylgenistein (2), two natural flavonoids with potential against Alzheimer’s disease (AD). Chrysin was used as the prototype structure for chemical modification in the present work. Full article ">Figure 2
Structure of the new chromones, flavones and their C-glucosyl derivatives studied in this work (for the synthetic approach followed, see <a href="#app1-pharmaceuticals-12-00098" class="html-app">Supplementary Materials</a>). Full article ">Figure 3
Neuroprotective effects of compound 2, flavone derivatives and corresponding aglycones against H2O2-induced toxicity in human SH-SY5Y neuroblastoma cells via a MTT cell viability assay. (A) Effects caused by amine moieties in para-position of ring B; (B) effects caused by the replacement of ring B with heteroaromatic groups; (C) effects caused by electron withdrawing groups in para-position of ring B; (D) effects caused by the replacement of ring B with aliphatic moieties. Cells were incubated with 100 μM H2O2 for 24 h at 37 °C, in the presence (50 μM) or absence of each compound. The tests were performed in triplicate with a final concentration of 0.5% DMSO. Results are presented as means ± standard error. Statistical differences between groups were assessed by one-way ANOVA followed by a Tukey’s post-test. * p < 0.05, and ** p < 0.01 versus cell control; & p < 0.05, && p < 0.01, &&& p < 0.001 and &&&& p < 0.0001 versus H2O2 control; §§ p < 0.01 and §§§ p < 0.001 versus another compound. Full article ">Figure 4
Neuroprotective effects of compound 2 and analogues against Aβ1-42-induced toxicity in human SH-SY5Y neuroblastoma cells via a MTT cell viability assay. Cells were incubated with 20 μM Aβ1-42 for 24 h at 37 °C, in the presence (50 μM) or absence of each compound. The tests were performed in triplicate with 1% DMSO (Aβ) or 1.5% DMSO (Aβ + compound − maximum DMSO percentage presented in the graph). Results are presented as means ± standard error. Statistical differences between groups were assessed by one-way ANOVA followed by a Tukey’s post-test. *** p < 0.001 and **** p < 0.0001 versus cell control; & p < 0.05, && p < 0.01 and &&&& p < 0.0001 Aβ control. Full article ">

11 pages, 497 KiB

Open AccessReview

Lipid Metabolism as a Source of Druggable Targets for Antiviral Discovery against Zika and Other Flaviviruses

by Miguel A. Martín-Acebes, Nereida Jiménez de Oya and Juan-Carlos Saiz

Pharmaceuticals 2019, 12(2), 97; https://doi.org/10.3390/ph12020097 - 21 Jun 2019

Cited by 38 | Viewed by 6904

Abstract

The Zika virus (ZIKV) is a mosquito-borne flavivirus that can lead to birth defects (microcephaly), ocular lesions and neurological disorders (Guillain-Barré syndrome). There is no licensed vaccine or antiviral treatment against ZIKV infection. The effort to understand the complex interactions of ZIKV with [...] Read more.

The Zika virus (ZIKV) is a mosquito-borne flavivirus that can lead to birth defects (microcephaly), ocular lesions and neurological disorders (Guillain-Barré syndrome). There is no licensed vaccine or antiviral treatment against ZIKV infection. The effort to understand the complex interactions of ZIKV with cellular networks contributes to the identification of novel host-directed antiviral (HDA) candidates. Among the cellular pathways involved in infection, lipid metabolism gains attention. In ZIKV-infected cells lipid metabolism attributed to intracellular membrane remodeling, virion morphogenesis, autophagy modulation, innate immunity and inflammation. The key roles played by the cellular structures associated with lipid metabolism, such as peroxisomes and lipid droplets, are starting to be deciphered. Consequently, there is a wide variety of lipid-related antiviral strategies that are currently under consideration, which include an inhibition of sterol regulatory element-binding proteins (SREBP), the activation of adenosine-monophosphate activated kinase (AMPK), an inhibition of acetyl-Coenzyme A carboxylase (ACC), interference with sphingolipid metabolism, blockage of intracellular cholesterol trafficking, or a treatment with cholesterol derivatives. Remarkably, most of the HDAs identified in these studies are also effective against flaviviruses other than ZIKV (West Nile virus and dengue virus), supporting their broad-spectrum effect. Considering that lipid metabolism is one of the main cellular pathways suitable for pharmacological intervention, the idea of repositioning drugs targeting lipid metabolism as antiviral candidates is gaining force. Full article

(This article belongs to the Special Issue Zika Virus: Therapeutic Advances)

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14 pages, 705 KiB

Open AccessReview

Ferritin in Kidney and Vascular Related Diseases: Novel Roles for an Old Player

by József Balla, György Balla and Abolfazl Zarjou

Pharmaceuticals 2019, 12(2), 96; https://doi.org/10.3390/ph12020096 - 21 Jun 2019

Cited by 23 | Viewed by 5351

Abstract

Iron is at the forefront of a number of pivotal biological processes due to its ability to readily accept and donate electrons. However, this property may also catalyze the generation of free radicals with ensuing cellular and tissue toxicity. Accordingly, throughout evolution numerous [...] Read more.

Iron is at the forefront of a number of pivotal biological processes due to its ability to readily accept and donate electrons. However, this property may also catalyze the generation of free radicals with ensuing cellular and tissue toxicity. Accordingly, throughout evolution numerous pathways and proteins have evolved to minimize the potential hazardous effects of iron cations and yet allow for readily available iron cations in a wide variety of fundamental metabolic processes. One of the extensively studied proteins in the context of systemic and cellular iron metabolisms is ferritin. While clinicians utilize serum ferritin to monitor body iron stores and inflammation, it is important to note that the vast majority of ferritin is located intracellularly. Intracellular ferritin is made of two different subunits (heavy and light chain) and plays an imperative role as a safe iron depot. In the past couple of decades our understanding of ferritin biology has remarkably improved. Additionally, a significant body of evidence has emerged describing the significance of the kidney in iron trafficking and homeostasis. Here, we briefly discuss some of the most important findings that relate to the role of iron and ferritin heavy chain in the context of kidney-related diseases and, in particular, vascular calcification, which is a frequent complication of chronic kidney disease. Full article

(This article belongs to the Special Issue Iron as Therapeutic Targets in Human Diseases)

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13 pages, 470 KiB

Open AccessReview

Therapeutic Potential of Kappa Opioid Agonists

by Tyler C. Beck, Matthew A. Hapstack, Kyle R. Beck and Thomas A. Dix

Pharmaceuticals 2019, 12(2), 95; https://doi.org/10.3390/ph12020095 - 20 Jun 2019

Cited by 55 | Viewed by 8934

Abstract

Many original research articles have been published that describe findings and outline areas for the development of kappa-opioid agonists (KOAs) as novel drugs; however, a single review article that summarizes the broad potential for KOAs in drug development does not exist. It is [...] Read more.

Many original research articles have been published that describe findings and outline areas for the development of kappa-opioid agonists (KOAs) as novel drugs; however, a single review article that summarizes the broad potential for KOAs in drug development does not exist. It is well-established that KOAs demonstrate efficacy in pain attenuation; however, KOAs also have proven to be beneficial in treating a variety of novel but often overlapping conditions including cardiovascular disease, pruritus, nausea, inflammatory diseases, spinal anesthesia, stroke, hypoxic pulmonary hypertension, multiple sclerosis, addiction, and post-traumatic cartilage degeneration. This article summarizes key findings of KOAs and discusses the untapped therapeutic potential of KOAs in the treatment of many human diseases. Full article

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10 pages, 655 KiB

Open AccessReview

Ironing out Macrophage Immunometabolism

by Stefania Recalcati, Elena Gammella and Gaetano Cairo

Pharmaceuticals 2019, 12(2), 94; https://doi.org/10.3390/ph12020094 - 19 Jun 2019

Cited by 26 | Viewed by 5026

Abstract

Over the last decade, increasing evidence has reinforced the key role of metabolic reprogramming in macrophage activation. In addition to supporting the specific immune response of different subsets of macrophages, intracellular metabolic pathways also directly control the specialized effector functions of immune cells. [...] Read more.

Over the last decade, increasing evidence has reinforced the key role of metabolic reprogramming in macrophage activation. In addition to supporting the specific immune response of different subsets of macrophages, intracellular metabolic pathways also directly control the specialized effector functions of immune cells. In this context, iron metabolism has been recognized as an important component of macrophage plasticity. Since macrophages control the availability of this essential metal, changes in the expression of genes coding for the major proteins of iron metabolism may result in different iron availability for the macrophage itself and for other cells in the microenvironment. In this review, we discuss how macrophage iron can also play a role in immunometabolism. Full article

(This article belongs to the Special Issue Iron as Therapeutic Targets in Human Diseases)

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11 pages, 498 KiB

Open AccessReview

Cellular Senescence and Iron Dyshomeostasis in Alzheimer’s Disease

by Shashank Masaldan, Abdel Ali Belaidi, Scott Ayton and Ashley I. Bush

Pharmaceuticals 2019, 12(2), 93; https://doi.org/10.3390/ph12020093 - 19 Jun 2019

Cited by 80 | Viewed by 7590

Abstract

Iron dyshomeostasis is a feature of Alzheimer’s disease (AD). The impact of iron on AD is attributed to its interactions with the central proteins of AD pathology (amyloid precursor protein and tau) and/or through the iron-mediated generation of prooxidant molecules (e.g., hydroxyl radicals). [...] Read more.

Iron dyshomeostasis is a feature of Alzheimer’s disease (AD). The impact of iron on AD is attributed to its interactions with the central proteins of AD pathology (amyloid precursor protein and tau) and/or through the iron-mediated generation of prooxidant molecules (e.g., hydroxyl radicals). However, the source of iron accumulation in pathologically relevant regions of the brain and its contribution to AD remains unclear. One likely contributor to iron accumulation is the age-associated increase in tissue-resident senescent cells that drive inflammation and contribute to various pathologies associated with advanced age. Iron accumulation predisposes ageing tissue to oxidative stress that can lead to cellular dysfunction and to iron-dependent cell death modalities (e.g., ferroptosis). Further, elevated brain iron is associated with the progression of AD and cognitive decline. Elevated brain iron presents a feature of AD that may be modified pharmacologically to mitigate the effects of age/senescence-associated iron dyshomeostasis and improve disease outcome. Full article

(This article belongs to the Special Issue Iron as Therapeutic Targets in Human Diseases)

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12 pages, 1351 KiB

Open AccessReview

CDK8-Novel Therapeutic Opportunities

by Ingeborg Menzl, Agnieszka Witalisz-Siepracka and Veronika Sexl

Pharmaceuticals 2019, 12(2), 92; https://doi.org/10.3390/ph12020092 - 19 Jun 2019

Cited by 34 | Viewed by 7735

Abstract

Improvements in cancer therapy frequently stem from the development of new small-molecule inhibitors, paralleled by the identification of biomarkers that can predict the treatment response. Recent evidence supports the idea that cyclin-dependent kinase 8 (CDK8) may represent a potential drug target for breast [...] Read more.

Improvements in cancer therapy frequently stem from the development of new small-molecule inhibitors, paralleled by the identification of biomarkers that can predict the treatment response. Recent evidence supports the idea that cyclin-dependent kinase 8 (CDK8) may represent a potential drug target for breast and prostate cancer, although no CDK8 inhibitors have entered the clinics. As the available inhibitors have been recently reviewed, we focus on the biological functions of CDK8 and provide an overview of the complexity of CDK8-dependent signaling throughout evolution and CDK8-dependent effects that may open novel treatment avenues. Full article

(This article belongs to the Special Issue Protein Kinases and Cancer)

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17 pages, 1732 KiB

Open AccessArticle

Synthesis and Study of New Quinolineaminoethanols as Anti-Bacterial Drugs

by Pierre Laumaillé, Alexandra Dassonville-Klimpt, François Peltier, Catherine Mullié, Claire Andréjak, Sophie Da-Nascimento, Sandrine Castelain and Pascal Sonnet

Pharmaceuticals 2019, 12(2), 91; https://doi.org/10.3390/ph12020091 - 18 Jun 2019

Cited by 2 | Viewed by 4119

Abstract

The lack of antibiotics with a novel mode of action associated with the spread of drug resistant bacteria make the fight against infectious diseases particularly challenging. A quinoline core is found in several anti-infectious drugs, such as mefloquine and bedaquiline. Two main objectives [...] Read more.

The lack of antibiotics with a novel mode of action associated with the spread of drug resistant bacteria make the fight against infectious diseases particularly challenging. A quinoline core is found in several anti-infectious drugs, such as mefloquine and bedaquiline. Two main objectives were set in this work. Firstly, we evaluated the anti-mycobacterial properties of the previous quinolines 3, which have been identified as good candidates against ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli) bacteria. Secondly, a new series 4 was designed and assessed against the same bacteria strains, taking the pair of enantiomers 3m/3n as the lead. More than twenty compounds 4 were prepared through a five-step asymmetric synthesis with good enantiomeric excesses (>90%). Interestingly, all compounds of series 3 were efficient on M. avium with MIC = 2–16 µg/mL, while series 4 was less active. Both series 3 and 4 were generally more active than mefloquine against the ESKAPEE bacteria. The quinolines 4 were either active against Gram-positive bacteria (MIC ≤ 4 µg/mL for 4c–4h and 4k/4l) or E. coli (MIC = 32–64 µg/mL for 4q–4v) according to the global lipophilicity of these compounds. Full article

(This article belongs to the Special Issue Selected Papers from the 4th International Electronic Conference on Medicinal Chemistry)

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15 pages, 2109 KiB

Open AccessArticle

Novel 11-Substituted Ellipticines as Potent Anticancer Agents with Divergent Activity against Cancer Cells

by Charlotte M. Miller, Elaine C. O’Sullivan and Florence O. McCarthy

Pharmaceuticals 2019, 12(2), 90; https://doi.org/10.3390/ph12020090 - 14 Jun 2019

Cited by 21 | Viewed by 4938

Abstract

Ellipticines have well documented anticancer activity, in particular with substitution at the 1-, 2-, 6- and 9-positions. However, due to limitations in synthesis and coherent screening methodology the full SAR profile of this anticancer class has not yet been achieved. In order to [...] Read more.

Ellipticines have well documented anticancer activity, in particular with substitution at the 1-, 2-, 6- and 9-positions. However, due to limitations in synthesis and coherent screening methodology the full SAR profile of this anticancer class has not yet been achieved. In order to address this shortfall, we have set out to explore the anticancer activity of this potent natural product by substitution. We currently describe the synthesis of novel 11-substituted ellipticines with two specific derivatives showing potency and diverging cellular growth effects. Full article

(This article belongs to the Special Issue Anticancer Drugs)

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22 pages, 3119 KiB

Open AccessArticle

CK2 Pro-Survival Role in Prostate Cancer Is Mediated via Maintenance and Promotion of Androgen Receptor and NFκB p65 Expression

by Janeen H. Trembley, Betsy T. Kren, Md. J. Abedin, Daniel P. Shaughnessy, Yingming Li, Scott M. Dehm and Khalil Ahmed

Pharmaceuticals 2019, 12(2), 89; https://doi.org/10.3390/ph12020089 - 14 Jun 2019

Cited by 14 | Viewed by 4824

Abstract

The prosurvival protein kinase CK2, androgen receptor (AR), and nuclear factor kappa B (NFκB) interact in the function of prostate cells, and there is evidence of crosstalk between these signals in the pathobiology of prostate cancer (PCa). As CK2 is elevated in PCa, [...] Read more.

The prosurvival protein kinase CK2, androgen receptor (AR), and nuclear factor kappa B (NFκB) interact in the function of prostate cells, and there is evidence of crosstalk between these signals in the pathobiology of prostate cancer (PCa). As CK2 is elevated in PCa, and AR and NFκB are involved in the development and progression of prostate cancer, we investigated their interaction in benign and malignant prostate cells in the presence of altered CK2 expression. Our results show that elevation of CK2 levels caused increased levels of AR and NFκB p65 in prostate cells of different phenotypes. Analysis of TCGA PCa data indicated that AR and CK2α RNA expression are strongly correlated. Small molecule inhibition or molecular down-regulation of CK2 caused reduction in AR mRNA expression and protein levels in PCa cells and in orthotopic xenograft tumors by various pathways. Among these, regulation of AR protein stability plays a unifying role in CK2 maintenance of AR protein levels. Our results show induction of various endoplasmic reticulum stress signals after CK2 inhibition, which may play a role in the PCa cell death response. Of note, CK2 inhibition caused loss of cell viability in both parental and enzalutamide-resistant castrate-resistant PCa cells. The present work elucidates the specific link of CK2 to the pathogenesis of PCa in association with AR and NFκB expression; further, the observation that inhibition of CK2 can exert a growth inhibitory effect on therapy-resistant PCa cells emphasizes the potential utility of CK2 inhibition in patients who are on enzalutamide treatment for advanced cancer. Full article

(This article belongs to the Special Issue Protein Kinases and Cancer)

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Figure 1
Effects of increased CK2α expression on AR and NFκB p65 protein levels. (A) Immunoblot analysis of RWPE-1 cells stably expressing Flag-CK2α after 45, 62 and 73 days in culture. Control lanes represent either parental RWPE-1 cells or RWPE-1 cells stably expressing empty vector collected simultaneously with Flag-CK2α cells. Proteins detected are indicated on the right side of the blots. Actin signal was used as the loading control. (B) Immunoblot analysis of RWPE-1 and C4-2B cell lysates 24 h after transient transfection with Flag-CK2α expression construct. Control lanes represent empty vector transfected cells. β-Tubulin signal was used as the loading control. Arrows indicate correct band. Full article ">Figure 2
Co-expression of CSNK2A1 and AR genes in prostate cancer patient primary tumor samples. Analysis of co-expression of CSNK2A1 and AR mRNA levels in PCa patient samples from The Cancer Genome Atlas (Pan-Cancer Atlas; n = 494 samples). Correlation analysis and p-values provided within panel. Analysis performed using cBioPortal with Z-score cut-off set at 1.5. RSEM, RNA-Seq by Expectation–Maximization. Full article ">Figure 3
Blocking CK2 expression and activity reduces AR protein levels in PCa cells. (A) LNCaP (left panels) and C4-2 (right panels) cells were transfected with CK2αα’-targeted and control siRNAs. Cells were collected 48 and 72 h post-transfection for analysis by immunoblot. (B) LNCaP, C4-2, and 22Rv1 cells were treated with 80 µM TBB, 10 µM CX-4945, or equivalent concentration of DMSO. Cells were collects at various time points, as labeled above the lanes, for analysis by immunoblot. UnT = untreated cells. Vertical lines indicate non-contiguous lanes. (C) C4-2B and 22Rv1 cells were treated with 20 µM TBB, 40 µM TBB, or equivalent concentration of DMSO. Cells were collected at 24 h for analysis by immunoblot. Mean and 95% confidence intervals for AR protein levels after TBB treatment relative to DMSO treatment are indicated below the AR bands. For all panels: Proteins detected are indicated on the right side of blots, time points analyzed are indicated below the blots, and either actin or β-tubulin were used as loading controls. Arrows indicate correct band. Full article ">Figure 4
Blocking CK2 expression and activity reduces NFκB p65 protein levels and activation in PCa cells. (A) LNCaP cells were transfected with CK2αα’-targeted and control siRNAs. Cells were collected 48 and 72 h post-transfection, as labeled below the blots, for analysis by immunoblot. (B) C4-2B and 22Rv1 cells were treated with 20 µM TBB, 40 µM TBB or equivalent concentration of DMSO as indicated. Cells were collected at 24 h for analysis by immunoblot. Vertical lines indicate non-contiguous lanes. For all panels: proteins detected are indicated on the right side of blots and either actin or β-tubulin were used as loading controls. Arrows indicate correct band. Means and 95% confidence intervals for values relative to siControl or DMSO are indicated below each blot. Full article ">Figure 5
Anti-CK2 nanocapsule RNAi-based systemic treatment reduces expression of CK2, AR and NFκB p65 in 22Rv1 orthotopic xenograft tumors. (A) Orthotopic 22Rv1 tumors were initiated in NOD SCID gamma castrated male mice. When tumors were palpable, mice were treated on days 1, 4 and 7 with TBG-RNAi-CK2 or TBG-RNAi-F7 (control) nanocapsule by tail vein injection (0.02 mg/kg). Tumors were harvested on day 8, 24 h after the last treatment. Tumors were weighed, dissected to remove dead tissue, and reweighed. Mean tumor weights per group are indicated on the left panel and the mean percent of dead tumor tissue removed is indicated on the right panel. TBG-RNAi-CK2, n = 4; TBG-RNAi-F7, n = 3. Error bars indicate standard error. (B) Orthotopic xenograft 22Rv1 tumors from mice treated with TBG-RNAi-CK2 or TBG-RNAi-F7 (control) nanocapsule drugs were subjected to immunoblot analysis. Treatments are indicated above the lanes, and proteins detected are indicated to the right of the panels. Mean expression levels and 95% confidence intervals are indicated below the individual blots. Actin was used as a loading control. Full article ">Figure 6
Androgen is not required for CK2 block-mediated loss of AR protein levels, and AR loss is equivalent in non-malignant RWPE-1 cells. (A) C4-2 and 22Rv1 cells grown under androgen-free conditions were treated with 80 µM TBB or equivalent concentration of DMSO. Cells were collected 48 h post-treatment for analysis by immunoblot. (B) RWPE-1 cells were treated with 80 µM TBB or equivalent concentration of DMSO or transfected with CK2αα’-targeted and control siRNAs. Cells were analyzed by immunoblot at the time points indicated below the panels. For all panels: treatment conditions are indicated above the lanes, and proteins detected indicated to the right of the panels, and actin was used as a loading control. Full article ">Figure 7
CK2 expression and activity influence AR protein half-life and ER stress signaling. (A) 22Rv1 and C4-2B cells were treated with 80 µM TBB or equivalent concentration of DMSO. C4-2B cells were also transfected with CK2αα′-targeted and control siRNAs. Twenty-four hours after TBB or DMSO treatment or 22 h post-transfection, cycloheximide was added to the culture plates, and cells were collected over time as indicated above the blot lanes. Cell lysates were analyzed by immunoblot for AR protein expression, and AR half-life was calculated by linear regression of the quantitated signals. (B) C4-2 and 22Rv1 sample lysates from the experiments presented in <a href="#pharmaceuticals-12-00089-f003" class="html-fig">Figure 3</a>B were analyzed for markers of ER stress and autophagy by immunoblot. (C) AR and CHOP signals were analyzed by immunoblot in C4-2 and 22Rv1 48 h lysates following treatment with TBB or CX-4945. Full article ">Figure 8
CK2 inhibition decreases cell viability and induces loss of mitochondrial membrane potential in enzalutamide-resistant C4-2B prostate cancer cells. (A) C4-2B parental and enzalutamide-resistant cells were treated with 2-fold dilution series of TBB, CX-4945 or equivalent concentrations of DMSO for 72 h. Cell viability was measured by MTS-based Aqueous One assay. Experiment was performed three times. Error bars indicate standard error. * p < 0.05; ** p < 0.01; *** p < 0.001; # p < 0.0001. (B) C4-2B parental and enzalutamide-resistant cells were treated with 50 µM TBB, or equivalent volume DMSO for 2 h. JC-1 was added to the cells 1 h after TBB or DMSO addition. CCCP treatment at 50 µM for the last 30 min of incubation was used as positive control. Experiment was performed three times. Error bars indicate standard error. *** p < 0.001; # p < 0.0001. Full article ">Figure 9
Cartoon summary of the influence of CK2 expression and activity on AR and NFκB p65 protein expression and prostate tumor cell survival as described in this work. Full article ">

1 pages, 547 KiB

Open AccessCorrection

Correction: Klingler, M., et al. Cholecystokinin-2 Receptor Targeting with Novel C-terminally Stabilized HYNIC-Minigastrin Analogs Radiolabeled with Technetium-99m. Pharmaceuticals 2019, 12, 13

by Maximilian Klingler, Christine Rangger, Dominik Summer, Piriya Kaeopookum, Clemens Decristoforo and Elisabeth von Guggenberg

Pharmaceuticals 2019, 12(2), 88; https://doi.org/10.3390/ph12020088 - 13 Jun 2019

Cited by 4 | Viewed by 2870

Abstract

In our paper [...] Full article

(This article belongs to the Special Issue Targets, Tracers and Translation – Novel Radiopharmaceuticals Boost Nuclear Medicine)

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14 pages, 528 KiB

Open AccessReview

Cysteine Cathepsin Protease Inhibition: An update on its Diagnostic, Prognostic and Therapeutic Potential in Cancer

by Surinder M. Soond, Maria V. Kozhevnikova, Paul A. Townsend and Andrey A. Zamyatnin, Jr.

Pharmaceuticals 2019, 12(2), 87; https://doi.org/10.3390/ph12020087 - 11 Jun 2019

Cited by 47 | Viewed by 6688

Abstract

In keeping with recent developments in basic research; the importance of the Cathepsins as targets in cancer therapy have taken on increasing importance and given rise to a number of key areas of interest in the clinical setting. In keeping with driving basic [...] Read more.

In keeping with recent developments in basic research; the importance of the Cathepsins as targets in cancer therapy have taken on increasing importance and given rise to a number of key areas of interest in the clinical setting. In keeping with driving basic research in this area in a translational direction; recent findings have given rise to a number of exciting developments in the areas of cancer diagnosis; prognosis and therapeutic development. As a fast-moving area of research; the focus of this review brings together the latest findings and highlights the translational significance of these developments. Full article

(This article belongs to the Special Issue Design of Enzyme Inhibitors as Potential Drugs)

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16 pages, 3141 KiB

Open AccessReview

Recent Developments in Peptidyl Diaryl Phoshonates as Inhibitors and Activity-Based Probes for Serine Proteases

by Marta Maślanka and Artur Mucha

Pharmaceuticals 2019, 12(2), 86; https://doi.org/10.3390/ph12020086 - 10 Jun 2019

Cited by 10 | Viewed by 4639

Abstract

This review presents current achievements in peptidyl diaryl phosphonates as covalent, specific mechanism-based inhibitors of serine proteases. Along three decades diaryl phosphonates have emerged as invaluable tools in fundamental and applicative studies involving these hydrolases. Such an impact has been promoted by advantageous [...] Read more.

This review presents current achievements in peptidyl diaryl phosphonates as covalent, specific mechanism-based inhibitors of serine proteases. Along three decades diaryl phosphonates have emerged as invaluable tools in fundamental and applicative studies involving these hydrolases. Such an impact has been promoted by advantageous features that characterize the phosphonate compounds and their use. First, the synthesis is versatile and allows comprehensive structural modification and diversification. Accordingly, reactivity and specificity of these bioactive molecules can be easily controlled by appropriate adjustments of the side chains and the leaving groups. Secondly, the phosphonates target exclusively serine proteases and leave other oxygen and sulfur nucleophiles intact. Synthetic accessibility, lack of toxicity, and promising pharmacokinetic properties make them good drug candidates. In consequence, the utility of peptidyl diaryl phosphonates continuously increases and involves novel enzymatic targets and innovative aspects of application. For example, conjugation of the structures of specific inhibitors with reporter groups has become a convenient approach to construct activity-based molecular probes capable of monitoring location and distribution of serine proteases. Full article

(This article belongs to the Special Issue Design of Enzyme Inhibitors as Potential Drugs)

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13 pages, 851 KiB

Open AccessCommunication

1,2-Dihydroxyxanthone: Effect on A375-C5 Melanoma Cell Growth Associated with Interference with THP-1 Human Macrophage Activity

by Viviana Silva, Fátima Cerqueira, Nair Nazareth, Rui Medeiros, Amélia Sarmento, Emília Sousa and Madalena Pinto

Pharmaceuticals 2019, 12(2), 85; https://doi.org/10.3390/ph12020085 - 4 Jun 2019

Cited by 10 | Viewed by 4170

Abstract

Xanthones have been suggested as prospective candidates for cancer treatment. 1,2- dihydroxyxanthone (1,2-DHX) is known to interfere with the growth of several cancer cell lines. We investigated the effects of 1,2-DHX on the growth of the A375-C5 melanoma cell line and THP-1 human [...] Read more.

Xanthones have been suggested as prospective candidates for cancer treatment. 1,2- dihydroxyxanthone (1,2-DHX) is known to interfere with the growth of several cancer cell lines. We investigated the effects of 1,2-DHX on the growth of the A375-C5 melanoma cell line and THP-1 human macrophage activity. 1,2-DHX showed a moderate growth inhibition of A375-C5 melanoma cells (concentration that causes a 50% inhibition of cell growth (GI₅₀) = 55.0 ± 2.3 µM), but strongly interfered with THP-1 human macrophage activity. Supernatants from lipopolysaccharide (LPS)-stimulated THP-1 macrophage cultures exposed to 1,2-DHX significantly increased growth inhibition of A375-C5 cells, when compared to supernatants from untreated LPS-stimulated macrophages or to direct treatment with 1,2-DHX only. 1,2-DHX decreased THP-1 secretion of interleukin-1β (IL-1β) and interleukin-10 (IL-10), but stimulated tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) production. This xanthone also inhibited nitric oxide (NO) production by RAW 264.7 murine macrophages, possibly through inhibition of inducible NO synthase production. In conclusion, these findings suggest a potential impact of 1,2-DHX in melanoma treatment, not only due to a direct effect on cancer cells but also by modulation of macrophage activity. Full article

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18 pages, 1899 KiB

Open AccessReview

Developments in Carbohydrate-Based Cancer Therapeutics

by Farzana Hossain and Peter R. Andreana

Pharmaceuticals 2019, 12(2), 84; https://doi.org/10.3390/ph12020084 - 4 Jun 2019

Cited by 94 | Viewed by 11704

Abstract

Cancer cells of diverse origins express extracellular tumor-specific carbohydrate antigens (TACAs) because of aberrant glycosylation. Overexpressed TACAs on the surface of tumor cells are considered biomarkers for cancer detection and have always been prioritized for the development of novel carbohydrate-based anti-cancer vaccines. In [...] Read more.

Cancer cells of diverse origins express extracellular tumor-specific carbohydrate antigens (TACAs) because of aberrant glycosylation. Overexpressed TACAs on the surface of tumor cells are considered biomarkers for cancer detection and have always been prioritized for the development of novel carbohydrate-based anti-cancer vaccines. In recent years, progress has been made in developing synthetic, carbohydrate-based antitumor vaccines to improve immune responses associated with targeting these specific antigens. Tumor cells also exhaust more energy for proliferation than normal cells, by consuming excessive amounts of glucose via overexpressed sugar binding or transporting receptors located in the cellular membrane. Furthermore, inspired by the Warburg effect, glycoconjugation strategies of anticancer drugs have gained considerable attention from the scientific community. This review highlights a small cohort of recent efforts which have been made in carbohydrate-based cancer treatments, including vaccine design and the development of glycoconjugate prodrugs, glycosidase inhibiting iminosugars, and early cancer diagnosis. Full article

(This article belongs to the Special Issue Carbohydrates 2018)

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11 pages, 1631 KiB

Open AccessArticle

Voltammetric Evaluation of Diclofenac Tablets Samples through Carbon Black-Based Electrodes

by Carlos Eduardo Peixoto da Cunha, Edson Silvio Batista Rodrigues, Morgana Fernandes Alecrim, Douglas Vieira Thomaz, Isaac Yves Lopes Macêdo, Luane Ferreira Garcia, Jerônimo Raimundo de Oliveira Neto, Emily Kussmaul Gonçalves Moreno, Nara Ballaminut and Eric de Souza Gil

Pharmaceuticals 2019, 12(2), 83; https://doi.org/10.3390/ph12020083 - 4 Jun 2019

Cited by 17 | Viewed by 4486

Abstract

Diclofenac (DIC) is a non-steroidal anti-inflammatory drug of wide use around the world. Electroanalytical methods display a high analytical potential for application in pharmaceutical samples but the drawbacks concerning electrode fouling and reproducibility are of major concern. Henceforth, the aim of this work [...] Read more.

Diclofenac (DIC) is a non-steroidal anti-inflammatory drug of wide use around the world. Electroanalytical methods display a high analytical potential for application in pharmaceutical samples but the drawbacks concerning electrode fouling and reproducibility are of major concern. Henceforth, the aim of this work was to propose the use of alternative low-cost carbon black (CB) and ionic liquid (IL) matrix to modify the surface of pencil graphite electrodes (PGE) in order to quantify DIC in raw materials, intermediates, and final products, as well as in stability assays of tablets. The proposed method using CB+IL/PGE displayed good recovery (99.4%) as well as limits of detection (LOD) of 0.08 µmol L-1 and limits of quantification (LOQ) of 0.28 µmol L⁻¹. CB+IL/PGE response was five times greater than the unmodified PGE. CB+IL-PGE stands as an interesting alternative for DIC assessment in different pharmaceutical samples. Full article

(This article belongs to the Special Issue Advanced Electrochemical Sensors in Drug Sensing)

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Figure 1
(A) 3D plot obtained for differential pulse voltammetric (DPV) assays at different pH solutions for 25 µmol L−1 Diclofenac (DIC); inset: the plot of peak current values vs. pH. (B) Linear plot of peak potential vs. pH unit and (C) the major ionic forms of DIC. Full article ">Figure 2
First (–––), second (• • •), and third (– • –) sequential cyclic voltammetry (CV) (A); DPV scans obtained at pencil graphite electrodes (PGE) without polishment prior to each assay (B); Square wave (SW) voltammograms, It as total current, If as forward current, and Ib as backward current (C); CV assays performed at different scan rates: 25 mV s−1 (• • •), 50 mV s−1 (- - -), 100 mV s−1 (– • –), 250 mV s−1 (– – –), 500 mV s−1 (–––) (Inset: linear plot of peak current vs. v1/2) (D). All for 25 µmol L−1 DIC in pH 5.0 0.1 M acetate (ACS) at PGE. The proposed electrochemical reaction mechanisms for DIC (E). Full article ">Figure 3
DPVs performed with bare PGE (----), IL/PGE (– – –), and CB+IL/PGE (–––) for 40 µmol L−1 DIC in pH 3.0 0.1 mol L−1 ACS (n = 3). Full article ">Figure 4
Calibration curve obtained from DPV assays for increasing DIC concentrations at CB+IL/PGE (A) and PGE (B) in pH 3.0 0.1 mol L−1 ACS. Full article ">Figure 5
(A) DPV of 50 µmol L−1 DIC in pH 3.0, 0.1 mol L−1 ACS (n = 3) solution with 1 mg of manitol, lactose, and hydroxipropil cellulose; (B) 50 µmol L−1 DIC in pH 3.0 0.1 mol L-1 ACS (n = 3) solution; (C) 1 mg of manitol, lactose, and hydroxipropil cellulose in pH 3.0 0.1 mol L−1 ACS (n = 3) solution. All analysis conducted at CB+IL/PGE. Full article ">Scheme 1
PGE and CB+IL/PGE preparation. Full article ">

17 pages, 1909 KiB

Open AccessArticle

De Novo Design and In Vitro Testing of Antimicrobial Peptides against Gram-Negative Bacteria

by Boris Vishnepolsky, George Zaalishvili, Margarita Karapetian, Tornike Nasrashvili, Nato Kuljanishvili, Andrei Gabrielian, Alex Rosenthal, Darrell E. Hurt, Michael Tartakovsky, Maya Grigolava and Malak Pirtskhalava

Pharmaceuticals 2019, 12(2), 82; https://doi.org/10.3390/ph12020082 - 3 Jun 2019

Cited by 47 | Viewed by 7160

Abstract

Antimicrobial peptides (AMPs) have been identified as a potentially new class of antibiotics to combat bacterial resistance to conventional drugs. The design of de novo AMPs with high therapeutic indexes, low cost of synthesis, high resistance to proteases and high bioavailability remains a [...] Read more.

Antimicrobial peptides (AMPs) have been identified as a potentially new class of antibiotics to combat bacterial resistance to conventional drugs. The design of de novo AMPs with high therapeutic indexes, low cost of synthesis, high resistance to proteases and high bioavailability remains a challenge. Such design requires computational modeling of antimicrobial properties. Currently, most computational methods cannot accurately calculate antimicrobial potency against particular strains of bacterial pathogens. We developed a tool for AMP prediction (Special Prediction (SP) tool) and made it available on our Web site (https://dbaasp.org/prediction). Based on this tool, a simple algorithm for the design of de novo AMPs (DSP) was created. We used DSP to design short peptides with high therapeutic indexes against gram-negative bacteria. The predicted peptides have been synthesized and tested in vitro against a panel of gram-negative bacteria, including drug resistant ones. Predicted activity against Escherichia coli ATCC 25922 was experimentally confirmed for 14 out of 15 peptides. Further improvements for designed peptides included the synthesis of D-enantiomers, which are traditionally used to increase resistance against proteases. One synthetic D-peptide (SP15D) possesses one of the lowest values of minimum inhibitory concentration (MIC) among all DBAASP database short peptides at the time of the submission of this article, while being highly stable against proteases and having a high therapeutic index. The mode of anti-bacterial action, assessed by fluorescence microscopy, shows that SP15D acts similarly to cell penetrating peptides. SP15D can be considered a promising candidate for the development of peptide antibiotics. We plan further exploratory studies with the SP tool, aiming at finding peptides which are active against other pathogenic organisms. Full article

(This article belongs to the Special Issue Selected Papers from the 4th International Electronic Conference on Medicinal Chemistry)

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12 pages, 1682 KiB

Open AccessArticle

Cardiolipin-Based Lipopolyplex Platform for the Delivery of Diverse Nucleic Acids into Gram-Negative Bacteria

by Federico Perche, Tony Le Gall, Tristan Montier, Chantal Pichon and Jean-Marc Malinge

Pharmaceuticals 2019, 12(2), 81; https://doi.org/10.3390/ph12020081 - 28 May 2019

Cited by 11 | Viewed by 4202

Abstract

Antibiotic resistance is a growing public health concern. Because only a few novel classes of antibiotics have been developed in the last 40 years, such as the class of oxazolidinones, new antibacterial strategies are urgently needed (Coates, A.R. et al., 2011). Nucleic acid-based [...] Read more.

Antibiotic resistance is a growing public health concern. Because only a few novel classes of antibiotics have been developed in the last 40 years, such as the class of oxazolidinones, new antibacterial strategies are urgently needed (Coates, A.R. et al., 2011). Nucleic acid-based antibiotics are a new type of antimicrobials. However, free nucleic acids cannot spontaneously cross the bacterial cell wall and membrane; consequently, their intracellular delivery into bacteria needs to be assisted. Here, we introduce an original lipopolyplex system named liposome polymer nucleic acid (LPN), capable of versatile nucleic acid delivery into bacteria. We characterized LPN formed with significant therapeutic nucleic acids: 11 nt antisense single-stranded (ss) DNA and double-stranded (ds) DNA of 15 and 95 base pairs (bp), 9 kbp plasmid DNA (pDNA), and 1000 nt ssRNA. All these complexes were efficiently internalized by two different bacterial species, i.e., Escherichia coli and Pseudomonas aeruginosa, as shown by flow cytometry. Consistent with intracellular delivery, LPN prepared with an antisense oligonucleotide and directed against an essential gene, induced specific and important bacterial growth inhibition likely leading to a bactericidal effect. Our findings indicate that LPN is a versatile platform for efficient delivery of diverse nucleic acids into Gram-negative bacteria. Full article

(This article belongs to the Special Issue New Tools for Medicinal Chemists)

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Two-step preparation of liposome polymer nucleic acid (LPN). Full article ">Figure 2
Characterization of LPN: (a) Gel retardation of nucleic acids either free or LPN-complexed. (b) Morphology of LPN–ASO complexes imaged by electron microscopy, scale bar represents 100 nm. Full article ">Figure 3
Nucleic acid delivery into bacteria. Delivery of LPN prepared with FITC-labeled nucleic acids was evaluated by flow cytometry after 2 h incubation at 37 °C with bacteria. Full article ">Figure 4
Antibacterial activity of LPN. The growth of E. coli (a) and P. aeruginosa (b) after treatment with LPN–ASO complexes (1 µM ASO) or ampicillin (AMP, 150 µg/mL) was monitored for 5 h at 37 °C. Full article ">Figure 5
Cytotoxicity of LPN towards eukaryotic cells. Cell viability of dendritic cells 24 h after transfection with LPN complexes using increasing concentrations of ASO. Full article ">Figure A1
Chemical structures of polymer and lipids. PEI (polyethylenimine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), NBD-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)). Full article ">

12 pages, 1853 KiB

Open AccessArticle

Synthetic Inhibitors of Snake Venom Enzymes: Thioesters Derived from 2-Sulfenyl Ethylacetate

by Isabel C. Henao Castañeda, Jaime A. Pereañez and Lina M. Preciado

Pharmaceuticals 2019, 12(2), 80; https://doi.org/10.3390/ph12020080 - 23 May 2019

Cited by 13 | Viewed by 4395

Abstract

Snakebite envenomings are a global public health issue. The therapy based on the administration of animal-derived antivenoms has limited efficacy against the venom-induced local tissue damage, which often leads to permanent disability. Therefore, there is a need to find inhibitors against toxins responsible [...] Read more.

Snakebite envenomings are a global public health issue. The therapy based on the administration of animal-derived antivenoms has limited efficacy against the venom-induced local tissue damage, which often leads to permanent disability. Therefore, there is a need to find inhibitors against toxins responsible for local damage. This work aimed to synthesize thioesters derived from 2-sulfenyl ethylacetate and to evaluate the inhibitory effects on two snake venom toxins. Ethyl 2-((4-chlorobenzoyl)thio)acetate (I), Ethyl 2-((3-nitrobenzoyl)thio)acetate (II) and Ethyl 2-((4-nitrobenzoyl)thio)acetate (III) were synthesized and spectroscopically characterized. Computational calculations were performed to support the study. The inhibitory capacity of compounds (I–III) was evaluated on a phospholipase A₂ (Cdcum6) isolated from the venom of the Colombian rattlesnake Crotalus durissus cumanensis and the P-I type metalloproteinase Batx-I isolated from Bothrops atrox. I–III inhibited PLA₂ with IC₅₀ values of 193.2, 305.4 and 132.7 µM, respectively. Otherwise, compounds II and III inhibited the proteolytic activity of Batx-I with IC₅₀ of 2774 and 1879 µM. Molecular docking studies show that inhibition of PLA₂ may be due to interactions of the studied compounds with amino acids in the catalytic site and the cofactor Ca²⁺. Probably, a blockage of the hydrophobic channel and some amino acids of the interfacial binding surface of PLA₂ may occur. Full article

(This article belongs to the Special Issue Design of Enzyme Inhibitors as Potential Drugs)

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General structure of studied compounds: Ethyl 2-((4-chlorobenzoyl)thio)acetate (I), Ethyl 2-((3-nitrobenzoyl)thio)acetate (II) and Ethyl 2-((4-nitrobenzoyl)thio)acetate (III). Full article ">Figure 2
IC50 curve showing inhibition of the PLA2 enzymatic activity by compounds I, II and III. Full article ">Figure 3
IC50 curve showing inhibition of the metalloproteinase proteolytic activity by compounds II and III. Full article ">Figure 4
Potential energy curve around the dihedral angle δ S-C-C=O for compounds I, II and III at B3LYP/6-31++G(d,p) level of approximation. Full article ">Figure 5
Docking results PLA2 with compounds I (A), II (B) and III (C). Full article ">Figure 6
Binding of compounds I, II and III to the substrate binding cleft of the PLA2. The red areas of the surface represent the acid regions; the white areas represent the neutral and the blue areas the basic regions. The blue sphere represents Ca2+. Compound I (yellow), II (green) and III (red). Full article ">

21 pages, 8639 KiB

Open AccessReview

Targeting MMP-9 in Diabetic Foot Ulcers

by Jeffrey I. Jones, Trung T. Nguyen, Zhihong Peng and Mayland Chang

Pharmaceuticals 2019, 12(2), 79; https://doi.org/10.3390/ph12020079 - 22 May 2019

Cited by 64 | Viewed by 9547

Abstract

Diabetic foot ulcers (DFUs) are significant complications of diabetes and an unmet medical need. Matrix metalloproteinases (MMPs) play important roles in the pathology of wounds and in the wound healing process. However, because of the challenge in distinguishing active MMPs from the two [...] Read more.

Diabetic foot ulcers (DFUs) are significant complications of diabetes and an unmet medical need. Matrix metalloproteinases (MMPs) play important roles in the pathology of wounds and in the wound healing process. However, because of the challenge in distinguishing active MMPs from the two catalytically inactive forms of MMPs and the clinical failure of broad-spectrum MMP inhibitors in cancer, MMPs have not been a target for treatment of DFUs until recently. This review covers the discovery of active MMP-9 as the biochemical culprit in the recalcitrance of diabetic wounds to healing and targeting this proteinase as a novel approach for the treatment of DFUs. Active MMP-8 and MMP-9 were observed in mouse and human diabetic wounds using a batimastat affinity resin and proteomics. MMP-9 was shown to play a detrimental role in diabetic wound healing, whereas MMP-8 was beneficial. A new class of selective MMP-9 inhibitors shows clinical promise for the treatment of DFUs. Full article

(This article belongs to the Special Issue Matrix Metalloproteinases)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Three forms of matrix metalloproteinases (MMPs), as demonstrated for MMP-2: inactive zymogen (pro-MMP), active MMP, and inactive tissue inhibitor of metalloproteinases (TIMP)-complexed MMP. Cleavage of the prodomain (red) allows access to the catalytic site, while TIMPs block access to inactivate the MMP. Reproduced from Nguyen et al. [<a href="#B13-pharmaceuticals-12-00079" class="html-bibr">13</a>]. Full article ">Figure 2
Structures of (A) the TAPI-2 affinity resin and (B) the batimastat affinity resin used to capture active MMPs and related ADAMs (a disintegrin and metalloproteinase). The portion of the structure based on TAPI-2 is indicated in red and that based on batimastat is shown in blue. (C) Recovery of representative active MMPs and ADAMs by the batimastat affinity resin. Mouse tissue homogenate was spiked with 10 pmol of proteinase and incubated with the resin. Recovery was determined from quantitation of the proteinase recovered after the clean-up procedure relative to the spiked amount; mean ± SD, n = 3. Full article ">Figure 3
Selective MMP-9 inhibition accelerates diabetic wound healing. (A) Wound healing after treatment with 100 μg/wound/day of (R,S)-ND-336, (R,S)-ND-322, or vehicle. Mean ± standard error of the mean (SEM); n = 8/group on days 7, 10, and 14; * p < 0.05, ** p < 0.01 by Mann–Whitney U test. (B) Representative images of the wounds. (C) In-situ zymography of wounds with DQ-Gelatin and DQ-Collagen indicates that (R,S)-ND-336 inhibits MMP-9 but not MMP-8, while (R,S)-ND-322 inhibits MMP-9 and poorly inhibits MMP-8. Reproduced from Gao et al [<a href="#B86-pharmaceuticals-12-00079" class="html-bibr">86</a>]. Full article ">Figure 4
Aclerastide does not accelerate wound healing in diabetic mice when given at a clinically relevant dosing regimen. (A) Structure of aclerastide. Mice were treated topically 1 day after wound infliction with (R,S)-ND-336 or aclerastide at 100 µg/wound/day for 14 days or vehicle (water). (B) Wound closure measurements indicate that (R,S)-ND-336 accelerates wound healing faster than aclerastide; mean ± SEM, n = 12, 9, and 9 mice for (R,S)-ND-336 or aclerastide on days 7, 10, and 14, respectively; n = 13, 10, and 10 mice for vehicle on days 7, 10, and 14, respectively; ** p < 0.01, *** p < 0.001 by Mann–Whitney U two-tailed test. (C) Representative images of the wounds. (D) Increased active MMP-9 levels are observed in aclerastide-treated wounds by the batimastat affinity resin coupled with proteomics; mean ± S.D., n = 3 mice per group per time point, * p < 0.05 by Student’s t two-tailed test. (E) Aclerastide treatment increases the detrimental MMP-9 activity in diabetic wounds by in-situ zymography with DQ-Gelatin, whereas MMP-9 activity was inhibited in (R,S)-ND-336-treated wounds. Adapted from Nguyen et al. [<a href="#B49-pharmaceuticals-12-00079" class="html-bibr">49</a>]. Full article ">Figure 5
The role of MMP-8 in diabetic wound healing. (A) Structure of selective MMP-8 inhibitor. (B,C) Topical treatment with selective MMP-8 inhibitor (250 μg/wound/day) delays healing in diabetic mice; mean ± SD, n = 12, 6, and 6 on days 7, 10, and 14, respectively; * p < 0.05. (D,E) Topical treatment with exogenous recombinant MMP-8 (1 μg/wound/day) accelerates diabetic wound healing; mean ± SEM, n = 20, 9, and 9 mice on days 7, 10, and 14, respectively, for the vehicle group; n = 20, 10, and 10 mice on days 7, 10, and 14, respectively, for the MMP-8 group; * p < 0.05. Adapted from Gooyit et al. [<a href="#B67-pharmaceuticals-12-00079" class="html-bibr">67</a>] and Gao et al. [<a href="#B86-pharmaceuticals-12-00079" class="html-bibr">86</a>]. Full article ">Figure 6
(R)-ND-336 accelerates wound healing faster than (R,S)-ND-336 in diabetic mice. Topical treatment with (R)-, or (R,S)-ND-336 at 50 μg/wound/day for 14 days or vehicle (water). (A) Wound healing; mean ± SEM, n = 7 mice/group/time point for vehicle, (R,S)-ND-336, n = 8/time point for (R)-ND-336, * p < 0.05, ** p < 0.01 by Mann−Whitney U two-tailed test. (B) Representative wound images. (C) In-situ zymography of the wounds with DQ-gelatin shows that (R)-ND-336 inhibits MMP-9 better than (R,S)-ND-336; n = 3 mice/group. The bottom row shows merged images with DAPI nuclear DNA staining, 40× lens (scale bars, 50 μm). (D) In-situ zymography with DQ-collagen indicates that (R,S)- and (R)-ND-336 do not inhibit MMP-8; n = 3 mice/group. Adapted from Nguyen et al. [<a href="#B48-pharmaceuticals-12-00079" class="html-bibr">48</a>]. Full article ">Figure 7
(R)-ND-336 is superior to becaplermin in accelerating wound healing in diabetic mice. Mice were treated topically 1 day after wound infliction (8 mm, Tegaderm-covered) with 5 μg or 50 μg/wound/day of becaplermin or (R)-ND-336, respectively, or vehicle (water) for 14 days. (A) Wound measurements show that (R)-ND-336 has better efficacy than becaplermin, mean ± SEM; n = 11, 8, and 8 for vehicle, n = 11, 7, and 7 for (R)-ND-336, n = 12, 9, and 9 for becaplermin on days 7, 10, and 14, respectively; * p < 0.05, ** p < 0.01 by Mann−Whitney U two-tailed test. (B) Representative wound images. (C) Analysis of the wounds with the affinity resin coupled to proteomics indicates significant decrease in active MMP-9 in becaplermin-treated animals. (D) In-situ zymography with DQ-gelatin shows that (R)-ND-336 inhibits MMP-9 activity in vivo, while becaplermin decreases MMP-9 activity but does not completely inhibit it. In-situ zymography with DQ-collagen shows that (R)-ND-336 and becaplermin do not inhibit MMP-8. Images were taken with a 40× lens (scale bars, 50 μm). Adapted from Nguyen et al. [<a href="#B48-pharmaceuticals-12-00079" class="html-bibr">48</a>]. Full article ">Figure 8
Validation of the target MMP-9 in debridement tissue from human patients with diabetic foot ulcers (DFUs). Measurements of (A) active MMP-8 and (B) active MMP-9 in human DFUs stratified by the Wagner grade (WG) using the batimastat affinity resin coupled with proteomics; mean ± SEM: * p < 0.05, ** p < 0.01 by Mann−Whitney U two-tailed test. Adapted from Nguyen at al. [<a href="#B48-pharmaceuticals-12-00079" class="html-bibr">48</a>]. Full article ">Figure 9
Mechanism of inhibition of (R)-ND-336. (A) (R)-ND-336 inhibits MMP-9 as a mechanism-based inhibitor, where Glu-404 at the active site abstracts a proton α to sulfone, resulting in the corresponding thiolate that coordinates with zinc ion as a tight-binding inhibitor, for which the reversal occurs very slowly. (B) (R)-ND-336 inhibits MMP-9 as a slow-binding inhibitor with a long residence time of 300 ± 1 min. The compound is a poor non-competitive inhibitor for MMP-8, for which the residence time is very short. Adapted from Nguyen et al. [<a href="#B48-pharmaceuticals-12-00079" class="html-bibr">48</a>]. Full article ">Figure 10
Schematic showing the progress of translational work to bring a selective MMP-9 inhibitor towards the clinic for treatment of DFUs. Full article ">

6 pages, 539 KiB

Open AccessBrief Report

Tegsedi (Inotersen): An Antisense Oligonucleotide Approved for the Treatment of Adult Patients with Hereditary Transthyretin Amyloidosis

by Luís Gales

Pharmaceuticals 2019, 12(2), 78; https://doi.org/10.3390/ph12020078 - 21 May 2019

Cited by 46 | Viewed by 8838

Abstract

Tegsedi (Inotersen) is a chemically modified antisense oligonucleotide that inhibits the hepatic production of transthyretin (TTR). Several single-point mutations in TTR destabilize its structure, leading to the aggregation and accumulation of amyloid deposits in the nervous system, heart, kidneys and eyes. In July [...] Read more.

Tegsedi (Inotersen) is a chemically modified antisense oligonucleotide that inhibits the hepatic production of transthyretin (TTR). Several single-point mutations in TTR destabilize its structure, leading to the aggregation and accumulation of amyloid deposits in the nervous system, heart, kidneys and eyes. In July 2018, Tegsedi was approved by the European Commission for use in adults with stage one and two polyneuropathies. Later on, in October 2018, the FDA and Health Canada also approved its use for the treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis (hATTR) in adults in the U.S. and Canada. Tegsedi was developed by Ionis Pharmaceuticals, the company that holds the global marketing license, together with its subsidiary Akcea Therapeutics. Full article

(This article belongs to the Special Issue The Story of Successful Drugs and Recent FDA-Approved Molecules)

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15 pages, 894 KiB

Open AccessReview

The Expanding Role of MT1-MMP in Cancer Progression

by Anna M. Knapinska and Gregg B. Fields

Pharmaceuticals 2019, 12(2), 77; https://doi.org/10.3390/ph12020077 - 20 May 2019

Cited by 49 | Viewed by 7127

Abstract

For over 20 years, membrane type 1 matrix metalloproteinase (MT1-MMP) has been recognized as a key component in cancer progression. Initially, the primary roles assigned to MT1-MMP were the activation of proMMP-2 and degradation of fibrillar collagen. Proteomics has revealed a great array [...] Read more.

For over 20 years, membrane type 1 matrix metalloproteinase (MT1-MMP) has been recognized as a key component in cancer progression. Initially, the primary roles assigned to MT1-MMP were the activation of proMMP-2 and degradation of fibrillar collagen. Proteomics has revealed a great array of MT1-MMP substrates, and MT1-MMP selective inhibitors have allowed for a more complete mapping of MT1-MMP biological functions. MT1-MMP has extensive sheddase activities, is both a positive and negative regulator of angiogenesis, can act intracellularly and as a transcription factor, and modulates immune responses. We presently examine the multi-faceted role of MT1-MMP in cancer, with a consideration of how the diversity of MT1-MMP behaviors impacts the application of MT1-MMP inhibitors. Full article

(This article belongs to the Special Issue Matrix Metalloproteinases)

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15 pages, 7162 KiB

Open AccessArticle

Magnetic Graphene Oxide Nanocarrier for Targeted Delivery of Cisplatin: A Perspective for Glioblastoma Treatment

by Sami A. Makharza, Giuseppe Cirillo, Orazio Vittorio, Emanuele Valli, Florida Voli, Annafranca Farfalla, Manuela Curcio, Francesca Iemma, Fiore Pasquale Nicoletta, Ahmed A. El-Gendy, Gerardo F. Goya and Silke Hampel

Pharmaceuticals 2019, 12(2), 76; https://doi.org/10.3390/ph12020076 - 18 May 2019

Cited by 35 | Viewed by 5647

Abstract

Selective vectorization of Cisplatin (CisPt) to Glioblastoma U87 cells was exploited by the fabrication of a hybrid nanocarrier composed of magnetic γ-Fe₂O₃ nanoparticles and nanographene oxide (NGO). The magnetic component, obtained by annealing magnetite Fe₃O₄ and characterized [...] Read more.

Selective vectorization of Cisplatin (CisPt) to Glioblastoma U87 cells was exploited by the fabrication of a hybrid nanocarrier composed of magnetic γ-Fe₂O₃ nanoparticles and nanographene oxide (NGO). The magnetic component, obtained by annealing magnetite Fe₃O₄ and characterized by XRD measurements, was combined with NGO sheets prepared via a modified Hummer’s method. The morphological and thermogravimetric analysis proved the effective binding of γ-Fe₂O₃ nanoparticles onto NGO layers. The magnetization measured under magnetic fields up to 7 Tesla at room temperature revealed superparamagnetic-like behavior with a maximum value of M_S = 15 emu/g and coercivity H_C ≈ 0 Oe within experimental error. The nanohybrid was found to possess high affinity towards CisPt, and a rather slow fractional release profile of 80% after 250 h. Negligible toxicity was observed for empty nanoparticles, while the retainment of CisPt anticancer activity upon loading into the carrier was observed, together with the possibility to spatially control the drug delivery at a target site. Full article

(This article belongs to the Special Issue Anticancer Drugs)

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28 pages, 3354 KiB

Open AccessReview

Iron Supplementation Therapy, A Friend and Foe of Mycobacterial Infections?

by Rafiou Agoro and Catherine Mura

Pharmaceuticals 2019, 12(2), 75; https://doi.org/10.3390/ph12020075 - 17 May 2019

Cited by 25 | Viewed by 7840

Abstract

Iron is an essential element that is required for oxygen transfer, redox, and metabolic activities in mammals and bacteria. Mycobacteria, some of the most prevalent infectious agents in the world, require iron as growth factor. Mycobacterial-infected hosts set up a series of defense [...] Read more.

Iron is an essential element that is required for oxygen transfer, redox, and metabolic activities in mammals and bacteria. Mycobacteria, some of the most prevalent infectious agents in the world, require iron as growth factor. Mycobacterial-infected hosts set up a series of defense mechanisms, including systemic iron restriction and cellular iron distribution, whereas mycobacteria have developed sophisticated strategies to acquire iron from their hosts and to protect themselves from iron’s harmful effects. Therefore, it is assumed that host iron and iron-binding proteins, and natural or synthetic chelators would be keys targets to inhibit mycobacterial proliferation and may have a therapeutic potential. Beyond this hypothesis, recent evidence indicates a host protective effect of iron against mycobacterial infections likely through promoting remodeled immune response. In this review, we discuss experimental procedures and clinical observations that highlight the role of the immune response against mycobacteria under various iron availability conditions. In addition, we discuss the clinical relevance of our knowledge regarding host susceptibility to mycobacteria in the context of iron availability and suggest future directions for research on the relationship between host iron and the immune response and the use of iron as a therapeutic agent. Full article

(This article belongs to the Special Issue Iron as Therapeutic Targets in Human Diseases)

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Figure 1
Schematic representation of the cell envelope of Mycobacterium tuberculosis (adapted from [<a href="#B2-pharmaceuticals-12-00075" class="html-bibr">2</a>]). The cell envelope of M. tuberculosis includes an inner cell membrane (CM), a network of peptidoglycan (PG) with covalently attached macromolecules: AG, PIM2, PIM6, LM, and Man-LAM; an outer membrane (OM) composed of mycolic acid (MA), attached to TDM, DAT, PAT, PDIM, and SL-1; and an outmost layer of polysaccharides and proteins. AG: arabinogalactan; DAT: diacyltrehalose; LM: lipomannan; Man-LAM: mannose-capped lipoarabinomannan; PAT: poliacyltrehalose; PDIM: phthiocerol dimycocerosate; PIM2: phospho-myo-inositol-dimannoside; PIM6: phospho-myo-inositol-hexamannoside; MmpL: mycobacterial membrane protein large; TDM: trehalose dimycolate; SL-1: sulphoglycolipid. Full article ">Figure 2
Series of reactive oxygen species generated in response to intracellular mycobacteria infection. Macrophages and neutrophils produce reactive oxygen species to kill intracellular microbes in a series of reaction which can be initiated by the conversion of NADPH to NADP + via the NADPH oxidase (NOX) and the production of superoxide O2− in presence of O2. O2− can enter in the phagosome and later in the mycobacteria through porins or crosses the outer membrane that is partially permeabilized by antimicrobial peptides. O2− is a substrate for superoxide dismutase (SodCI, SodA) to produce H2O2 which can freely diffuse across membranes. The hydroxyl radical (OH•) is produced from H2O2 and superoxide via Fenton reaction. Mycobacteria produce the catalase KatG to detoxify H2O2. The production of NO from arginine metabolism via nitric oxide synthase (NOS2) can generate nitric oxide which in presence of O2− produce ONOO− and NO2−, which present some toxic proprieties against mycobacteria. Full article ">Figure 3
Structure of exochelin siderophore of M. smegmatis with its iron chelation proprieties. The iron-chelating residues of exochelin with their interaction with iron molecule is shown in red circle (adapted from [<a href="#B29-pharmaceuticals-12-00075" class="html-bibr">29</a>]). Full article ">Figure 4
Structure of mycobactin and carboxymycobactin siderophores of pathogenic mycobacteria. This figure represents mycobactin and carboxymycobactin from M. tuberculosis with the iron-chelating residues circled in red. Full article ">Figure 5
Heme-iron and siderophore-mediated iron acquisition of Mycobacterium tuberculosis in macrophage phagosome (adapted from [<a href="#B55-pharmaceuticals-12-00075" class="html-bibr">55</a>]). M. tuberculosis has both heme and non-heme iron uptake pathways. M. tuberculosis secretes and express on its outer membrane PPE36/37 which can bind to heme and induce heme importation across the outer membrane. Then heme is linked to Rv0265c which facilitate heme transport to the cytosol using an unknown heme importer. In the cytosol, heme is degraded by the heme-degrading protein MhuD and iron is extracted from heme. It is also possible than heme can also be exported from cytosol through MmpL3/11 transporters and then linked to Rv0203 and later exported outside the bacteria. M. tuberculosis can also acquire iron through the siderophore-mediated iron acquisition system. M. tuberculosis desferricarboxymycobactin competes with host iron transferrin to acquire iron in phagosome. Iron is then bound to mycobactin which transports iron across the inner membrane through the IrtAB transporter complex. Once in the cytosol, iron is dissociated from mycobactin which is then exported from the cytosol through MmpS4/L4 and MmpS5/L5 complex. Full article ">Figure 6
Mycobacterial stress sensors (adapted from [<a href="#B75-pharmaceuticals-12-00075" class="html-bibr">75</a>]) Mammalian host cell generates radicals and gases as a mechanism to counter mycobacterial infection. M. tuberculosis senses these potentially damaging stresses and responds by adjusting its energy metabolism, physiology and signaling and counters the damage by detoxification processes. M. tuberculosis possesses sensing mechanisms such as the Dos dormancy regulon and the WhiB3 redox sensor detecting environmental gases and alterations in its intracellular redox state. The Dos regulon senses O2, NO, and CO through the DosS and DosT heme proteins which transduce the signal to DosR leading to the induction of 48-member Dos dormancy regulon. WhiB3 functions as a regulator of cellular metabolism, which responds to O2 and NO through its Fe–S cluster and integrates it with intermediary metabolic pathways. WhiB3 is an intracellular redox regulator that dissipates reductive stress generated by utilization of host fatty acids through β-oxidation. Full article ">Figure 7
Suggested model of biphasic effect of iron supplementation during mycobacterial infection. A moderate concentration of iron supplementation can have a benefit effect for host during mycobacterial infection (shown in green). However, at high concentration of iron supplementation, iron can be beneficial for mycobacteria promoting growth and virulence (shown in red). Full article ">

12 pages, 945 KiB

Open AccessArticle

Retinal Gene Distribution and Functionality Implicated in Inherited Retinal Degenerations Can Reveal Disease-Relevant Pathways for Pharmacologic Intervention

by Debarshi Mustafi, Amirmohsen Arbabi, Hossein Ameri and Krzysztof Palczewski

Pharmaceuticals 2019, 12(2), 74; https://doi.org/10.3390/ph12020074 - 17 May 2019

Cited by 6 | Viewed by 4727

Abstract

The advent of genetic therapies for inherited retinal diseases (IRDs) has spurred the need for precise diagnosis and understanding of pathways for therapeutic targeting. The majority of IRDs that are clinically diagnosed, however, lack an identifiable mutation in established disease-causing loci and thus [...] Read more.

The advent of genetic therapies for inherited retinal diseases (IRDs) has spurred the need for precise diagnosis and understanding of pathways for therapeutic targeting. The majority of IRDs that are clinically diagnosed, however, lack an identifiable mutation in established disease-causing loci and thus can be investigated with limited rational drug discovery methods. Transcriptome profiling of the retina can reveal the functional state of the tissue, and geographic profiling can uncover the various clinical phenotypic presentations of IRDs and aid in pharmaceutical intervention. In this investigation, we detail the retinal geographic expression of known retinal disease-causing genes in the primate retina and functional targetable pathways in specific IRDs. Understanding the genetic basis as well as the resulting functional consequences will assist in the discovery of future therapeutic interventions and provide novel insights to medicinal chemists. Herein, we report that, despite the genetic heterogeneity of retinal diseases, potential functional pathways can be elucidated for therapeutic targeting and be used for predictive phenotypic and genotypic modeling of novel IRD presentations. Full article

(This article belongs to the Special Issue New Tools for Medicinal Chemists)

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42 pages, 12024 KiB

Open AccessMeeting Report

26th Annual GP2A Medicinal Chemistry Conference & 32nd Journées Franco-Belges de Pharmacochimie

by Patrick Dallemagne, Christophe Rochais, Pascal Marchand and Thierry Besson

Pharmaceuticals 2019, 12(2), 73; https://doi.org/10.3390/ph12020073 - 16 May 2019

Viewed by 7545

Abstract

As a joint meeting, the 26th Medicinal Chemistry Conference of GP2A and 32nd Journées Franco-Belges de Pharmacochimie took place between 13th and 15th June at Asnelles sur Mer (Normandie, France), providing a unique opportunity for a wide group of European medicinal chemists to [...] Read more.

As a joint meeting, the 26th Medicinal Chemistry Conference of GP2A and 32nd Journées Franco-Belges de Pharmacochimie took place between 13th and 15th June at Asnelles sur Mer (Normandie, France), providing a unique opportunity for a wide group of European medicinal chemists to engage. Topics included chemical tools for medicinal chemistry, protein-protein interactions, epigenetics, natural product-inspired molecules, computer-aided drug design, and new strategies for the design and development of drugs. Abstracts of invited lectures, proffered young researcher communications, flash communications and posters presented during the meeting are collected in this report. Full article

(This article belongs to the Special Issue Recent Contributions in Medicinal Chemistry Within European GP2A Network)

13 pages, 2120 KiB

Open AccessArticle

Colloidal Silver Induces Cytoskeleton Reorganization and E-Cadherin Recruitment at Cell-Cell Contacts in HaCaT Cells

by Elena Montano, Maria Vivo, Andrea Maria Guarino, Orsola di Martino, Blanda Di Luccia, Viola Calabrò, Sergio Caserta and Alessandra Pollice

Pharmaceuticals 2019, 12(2), 72; https://doi.org/10.3390/ph12020072 - 15 May 2019

Cited by 13 | Viewed by 12676

Abstract

Up until the first half of the 20th century, silver found significant employment in medical applications, particularly in the healing of open wounds, thanks to its antibacterial and antifungal properties. Wound repair is a complex and dynamic biological process regulated by several pathways [...] Read more.

Up until the first half of the 20th century, silver found significant employment in medical applications, particularly in the healing of open wounds, thanks to its antibacterial and antifungal properties. Wound repair is a complex and dynamic biological process regulated by several pathways that cooperate to restore tissue integrity and homeostasis. To facilitate healing, injuries need to be promptly treated. Recently, the interest in alternatives to antibiotics has been raised given the widespread phenomenon of antibiotic resistance. Among these alternatives, the use of silver appears to be a valid option, so a resurgence in its use has been recently observed. In particular, in contrast to ionic silver, colloidal silver, a suspension of metallic silver particles, shows antibacterial activity displaying less or no toxicity. However, the human health risks associated with exposure to silver nanoparticles (NP) appear to be conflicted, and some studies have suggested that it could be toxic in different cellular contexts. These potentially harmful effects of silver NP depend on various parameters including NP size, which commonly range from 1 to 100 nm. In this study, we analyzed the effect of a colloidal silver preparation composed of very small and homogeneous nanoparticles of 0.62 nm size, smaller than those previously tested. We found no adverse effect on the cell proliferation of HaCaT cells, even at high NP concentration. Time-lapse microscopy and indirect immunofluorescence experiments demonstrated that this preparation of colloidal silver strongly increased cell migration, re-modeled the cytoskeleton, and caused recruitment of E-cadherin at cell-cell junctions of human cultured keratinocytes. Full article

(This article belongs to the Special Issue Metal-Based Drugs: Updates and Perspectives)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Effects of AgC on HaCaT cell viability. MTT assay of HaCaT cells incubated for 24 or 48 h with colloidal (grey bars) or ionic silver (dark bars) at 0.5 or 2 μg/mL. Data are expressed as absorbance at 570 nm and presented as mean ± SE of three independent experiments, each done in sestuplicate. Analysis of variance was performed by two-way Anova followed by the Bonferroni post-test. **** P < 0.0001 when compared with the control. Full article ">Figure 2
Effect of AgC on stress granules formation. HaCaT cells were seeded on a coverslip and treated (or not) with AgC at 0.5 or 2 μg/mL for 24 h. Cells were then fixed and analyzed by TRITC-conjugated phalloidin staining (red) or indirect IF with the anti-YB1 antibody (green). Nuclei were stained with DAPI. Scale bar, 7 μM. Images were acquired using a Nikon TE Eclipse 2000. Full article ">Figure 3
Effect of AgC on wound closure dynamics. (A) Representative phase contrast microscopy images of cells incubated (or not) with AgC at different time points showing the wound closure process over time. (B,C) The wound closure process was quantified by measuring the reduction of the wound area (A) over time, as described in the Materials and Methods Section. Evolution of the wound area A, normalized to the value A0 (A at time 0), is reported for the control (B) and AgC treated (C) cells by selecting random fields along the wound for each experiment. The linear range of each data series was fit in order to measure the wound closure velocity α. Values of α for the control and treated cells are indicated on each graph as the mean from three independent experiments analyzed in triplicate. Standard error of the mean was calculated to account for reproducibility, and the t-test was calculated to verify the statistical significance of the differences with respect to the control samples. Full article ">Figure 4
Effect of AgC on wound closure dynamics. Values of α (wound closure velocity) (A), τ duplication time) (B), and D (cell motility coefficient) (C) of the control and AgC treated cells are reported. Values of D were calculated according to the Fisher–Kolmogoroff equation from values of α and τ (see Materials and Methods). Data are expressed as the mean of at least three independent experiments. SEM is reported as error bars, statistical significance was assessed by the paired two-tailed t-test (* P = 0.04; ** P = 0.007). Full article ">Figure 5
E-cadherin localization in HaCaT cells upon AgC treatment. Cells were allowed to adhere onto coverslips for 24 h and then treated with 0.5 μg/mL AgC for 8 h. Cells were then fixed and subjected to IF with an anti-E-cadherin antibody followed by TRITC-conjugated phalloidin to visualize the actin cytoskeleton. Representative images of E-cadherin subcellular localization and phalloidin are shown. Merged images also show DAPI staining to visualize the nuclei. Images were taken with a Zeiss confocal laser-scanning microscope Axio Observer (scale bar, 15 μM). A ×40 objective was used and image analysis was performed using ImageJ. Full article ">

18 pages, 1213 KiB

Open AccessArticle

VitalSign⁶: A Primary Care First (PCP-First) Model for Universal Screening and Measurement-Based Care for Depression

by Madhukar H. Trivedi, Manish K. Jha, Farra Kahalnik, Ronny Pipes, Sara Levinson, Tiffany Lawson, A. John Rush, Joseph M. Trombello, Bruce Grannemann, Corey Tovian, Robert Kinney, E. Will Clark and Tracy L. Greer

Pharmaceuticals 2019, 12(2), 71; https://doi.org/10.3390/ph12020071 - 14 May 2019

Cited by 40 | Viewed by 6948

Abstract

Major depressive disorder affects one in five adults in the United States. While practice guidelines recommend universal screening for depression in primary care settings, clinical outcomes suffer in the absence of optimal models to manage those who screen positive for depression. The current [...] Read more.

Major depressive disorder affects one in five adults in the United States. While practice guidelines recommend universal screening for depression in primary care settings, clinical outcomes suffer in the absence of optimal models to manage those who screen positive for depression. The current practice of employing additional mental health professionals perpetuates the assumption that primary care providers (PCP) cannot effectively manage depression, which is not feasible, due to the added costs and shortage of mental health professionals. We have extended our previous work, which demonstrated similar treatment outcomes for depression in primary care and psychiatric settings, using measurement-based care (MBC) by developing a model, called Primary Care First (PCP-First), that empowers PCPs to effectively manage depression in their patients. This model incorporates health information technology tools, through an electronic health records (EHR) integrated web-application and facilitates the following five components: (1) Screening (2) diagnosis (3) treatment selection (4) treatment implementation and (5) treatment revision. We have implemented this model as part of a quality improvement project, called VitalSign⁶, and will measure its success using the Reach, Efficacy, Adoption, Implementation, and Maintenance (RE-AIM) framework. In this report, we provide the background and rationale of the PCP-First model and the operationalization of VitalSign⁶ project. Full article

(This article belongs to the Special Issue Antidepressants: Mechanistic Insights and Future Directions)

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15 pages, 4360 KiB

Open AccessCommunication

Deregulation of Hepatic Mek1/2–Erk1/2 Signaling Module in Iron Overload Conditions

by Naveen Kumar Tangudu, Nils Buth, Pavel Strnad, Ion C. Cirstea and Maja Vujić Spasić

Pharmaceuticals 2019, 12(2), 70; https://doi.org/10.3390/ph12020070 - 7 May 2019

Cited by 46 | Viewed by 4415

Abstract

The liver, through the production of iron hormone hepcidin, controls body iron levels. High liver iron levels and deregulated hepcidin expression are commonly observed in many liver diseases including highly prevalent genetic iron overload disorders. In spite of a number of breakthrough investigations [...] Read more.

The liver, through the production of iron hormone hepcidin, controls body iron levels. High liver iron levels and deregulated hepcidin expression are commonly observed in many liver diseases including highly prevalent genetic iron overload disorders. In spite of a number of breakthrough investigations into the signals that control hepcidin expression, little progress has been made towards investigations into intracellular signaling in the liver under excess of iron. This study examined hepatic signaling pathways underlying acquired and genetic iron overload conditions. Our data demonstrate that hepatic iron overload associates with a decline in the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) kinase (Mek1/2) pathway by selectively affecting the phosphorylation of Erk1/2. We propose that Mek1/2-Erk1/2 signaling is uncoupled from iron-Bmp-Smad-mediated hepcidin induction and that it may contribute to a number of liver pathologies in addition to toxic effects of iron. We believe that our findings will advance the understanding of cellular signaling events in the liver during iron overload of different etiologies. Full article

(This article belongs to the Special Issue Iron as Therapeutic Targets in Human Diseases)

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Figure 1
Identification of activated proteins in iron overloaded livers by Bio-Plex Pro Cell Signaling MAPK Panel 9-plex. Phosphorylation status of nine intracellular phosphoproteins was measured in the livers of iron-dextran injected mice and compared to controls. Data were analyzed using GraphPad Prism software and results are shown as mean ± SEM (standard error of mean). For the statistical analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used. * p-values < 0.05; AU: arbitrary units; n = 4; 4 per group. Full article ">Figure 2
(a) Immunoblot analysis and relative quantification (shown in histograms on right) of pMEK1, pERK1/2, pStat3, and pp90Rsk in the livers of iron-dextran injected and control mice (n = 6; 6 mice per group). (b) Liver iron content (LIC) of control and iron-dextran injected mice. (c) Correlation analysis between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3, and pp90Rsk/pRsk1/2 in the livers of iron-dextran injected and control mice. M: Page Ruler Plus Prestained Protein Ladder (Thermo Scientific). Data were analyzed using GraphPad Prism software and results are shown as mean ± SEM (standard error of mean). For the statistical analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used. Linear regression and Pearson correlation coefficients were computed for every data set with 95% confidence intervals. * p values < 0.05. Full article ">Figure 3
(a) Representative immunoblot analysis and relative quantification (shown in histograms on right) of pMek1/2, pErk1/2, pStat3, and pp90Rsk in the livers of mice maintained on an iron rich diet (IRD) and standard diet (control) (n = 3–4 mice per group). (b) Liver iron content (LIC) of control and of mice maintained on an iron rich diet (n = 3–4 mice per group). (c) Correlation analysis between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3, and pp90Rsk/pRsk1/2 in the livers of mice maintained on an iron rich diet (IRD) and standard diet (control) (n = 3–4 mice per group). M: Page Ruler Plus Prestained Protein Ladder (Thermo Scientific). Data were analyzed using GraphPad Prism software and results are shown as mean ± SEM (standard error of mean). For the statistical analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used. Linear regression and Pearson correlation coefficients were computed for every data set with 95% confidence intervals. * p-values < 0.05. Full article ">Figure 4
(a) Representative immunoblot analysis and relative quantification (shown in histograms on right) of pMek1/2, pErk1/2, pStat3 and pp90Rsk in the livers of Hfe+/+ control and Hfe-/- mutant mice (n = 8;10 mice per group). (b) Liver iron content (LIC) of Hfe+/+ and Hfe-/- mice (n = 4;4 mice per group). (c) Correlation analysis between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3, and pp90Rsk/pRsk1/2 in the livers of Hfe+/+ and Hfe-/- mice (n = 4;4 mice per group). M: Page Ruler Plus Prestained Protein Ladder (Thermo Scientific). Data were analyzed using GraphPad Prism software and results are shown as mean ± SEM (standard error of mean). For the statistical analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used. Linear regression and Pearson correlation coefficients were computed for every data set with 95% confidence intervals. * p-values <0.05. Full article ">Figure 5
Immunoblot analysis and relative quantification (shown in histograms on right) of pMek1/2, pErk1/2, pStat3 and pp90Rsk in the livers of (a) control and Hamp-/- mice and (d) Hamp-/- mice maintained on an iron-rich diet (IRD) (n = 3;4;4 mice per group). Liver iron content (LIC) in (b) control, Hamp-/- and (e) Hamp-/- mice maintained on an iron-rich diet (n = 3;4;4 mice per group). Correlation analysis between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3, and pp90Rsk/pRsk1/2 in the livers of (c) control, Hamp-/- and (f) Hamp-/- mice maintained on an iron rich diet (IRD) (n = 3; 4;4 mice per group). M: Page Ruler Plus Prestained Protein Ladder (Thermo Scientific). Data were analyzed using GraphPad Prism software and results are shown as mean ± SEM (standard error of mean). For the statistical analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used. Linear regression and Pearson correlation coefficients were computed for every data set with 95% confidence intervals. * p-values <0.05. Full article ">Figure 5 Cont.
Immunoblot analysis and relative quantification (shown in histograms on right) of pMek1/2, pErk1/2, pStat3 and pp90Rsk in the livers of (a) control and Hamp-/- mice and (d) Hamp-/- mice maintained on an iron-rich diet (IRD) (n = 3;4;4 mice per group). Liver iron content (LIC) in (b) control, Hamp-/- and (e) Hamp-/- mice maintained on an iron-rich diet (n = 3;4;4 mice per group). Correlation analysis between LIC and the levels of pMek1/2/Mek1/2, pErk1/2/Erk1/2, pStat3/Stat3, and pp90Rsk/pRsk1/2 in the livers of (c) control, Hamp-/- and (f) Hamp-/- mice maintained on an iron rich diet (IRD) (n = 3; 4;4 mice per group). M: Page Ruler Plus Prestained Protein Ladder (Thermo Scientific). Data were analyzed using GraphPad Prism software and results are shown as mean ± SEM (standard error of mean). For the statistical analysis, a nonparametric distribution and the unpaired Mann–Whitney U Test were used. Linear regression and Pearson correlation coefficients were computed for every data set with 95% confidence intervals. * p-values <0.05. Full article ">Figure 6
Proposed model of two signaling pathways operating in iron-loaded livers. Systemic iron overload results in heavy iron deposition in the liver, illustrated here in form of high ferritin. Under these conditions, high levels of circulating transferrin-bound iron is sensed by cell membrane multiprotein iron-sensing complex, resulting in the activation of intracellular Bmp-Smad signaling cascade and increased hepcidin transcription in the nucleus. Independent of Bmp-Smad-mediated hepcidin activation, through a yet unknown mechanism (proposed here by red arrows), a decrease in phosphorylation of Mek1/2-Erk1/2-Stat3 (indicated in blue) occurs which in turn may affect the property of pErk1/2 and pStat3 signaling molecules to regulate gene transcription, alone or in cooperation with other transcription factors (TFs). A decrease in the activity of Mek1/2-Erk1/2 signaling cascade can be considered as liver response to iron overload. We propose that a decrease in Mek1/2-Erk1/2 signaling activity may accelerate liver pathologies in addition to toxic effects of iron. Full article ">

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Pharmaceuticals, Volume 12, Issue 2 (June 2019) – 54 articles

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