Novel Inhibitors of the Pseudomonas aeruginosa Virulence Factor LasB: a Potential Therapeutic Approach for the Attenuation of Virulence Mechanisms in Pseudomonal Infection

Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial bio?lm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed “second-generation” antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB,N-mercaptoacetyl-Phe-Tyr-amide (Ki 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in bio?lm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal bio?lms, and to eradicate bio?lm completely when used in combination with conventional antibiotics.

Binding modes of diketo-acid inhibitors of HIV-1 integrase: A comparative molecular dynamics simulation study

HIV-1 integrase (IN) has become an attractive target since drug resistance against HIV-1 reverse transcriptase (RT) and protease (PR) has appeared. Diketo acid (DKA) inhibitors are potent and selective inhibitors of HIV-1 IN: however the action mechanism is not well understood. Here, to study the inhibition mechanism of DKAs we performed 10 ns comparative molecular dynamics simulations on HIV-1 IN bound with three most representative DMA inhibitors: Shionogi inhibitor, S-1360 and two Merck inhibitors L-731,988 and L-708,906. Our simulations show that the acidic part of S-1360 formed salt bridge and cation-pi interactions with Lys159. In addition, the catalytic Glu152 in S-1360 was pushed away from the active site to form an ion-pair interaction with Arg199. The Merck inhibitors can maintain either one or both of these ion-pair interaction features. The difference in potencies of the DMA inhibitors is thus attributed to the different binding modes at the catalytic site. Such structural information at atomic level, not only demonstrates the action modes of DMA inhibitors but also provides a novel starting point for structural-based design of HIV-1 IN inhibitors.

Short Isoforms of the Cold Receptor TRPM8 Inhibit Channel Gating by Mimicking Heat Action Rather than Chemical Inhibitors

Transient receptor potential (TRP) channels couple various environmental factors to changes in membrane potential, calcium influx, and cell signaling. They also integrate multiple stimuli through their typically polymodal activation. Thus, although the TRPM8 channel has been extensively investigated as the major neuronal cold sensor, it is also regulated by various chemicals, as well as by several short channel isoforms. Mechanistic understanding of such complex regulation is facilitated by quantitative single-channel analysis. We have recently proposed a single-channel mechanism of TRPM8 regulation by voltage and temperature. Using this gating mechanism, we now investigate TRPM8 inhibition in cell-attached patches using HEK293 cells expressing TRPM8 alone or coexpressed with its short sM8-6 isoform. This is compared with inhibition by the chemicals N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC) and clotrimazole or by elevated temperature. We found that within the seven-state single-channel gating mechanism, inhibition of TRPM8 by short sM8-6 isoforms closely resembles inhibition by increased temperature. In contrast, inhibition by BCTC and that by clotrimazole share a different set of common features. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Proton pump inhibitors and histamine-2-receptor antagonists and pancreatic cancer risk: a nested case-control study

Background: The relationship between use of proton pump inhibitors (PPIs) and histamine-2-receptor antagonists (H₂RAs) and pancreatic cancer risk has yet to be examined. Data from a range of studies suggest biologically plausible mechanisms, whereby these drugs (or the conditions for which they are prescribed) may affect pancreatic cancer risk. The objective of this study was to investigate the relationship between use of PPIs/H₂RAs and pancreatic cancer risk.

Methods: A nested case – control study was conducted within the UK general practice research database (GPRD). Cases had a diagnosis of exocrine pancreatic cancer and controls were matched to cases on general practice site, sex and year of birth. Exposure to PPIs and to H₂RAs since entry into GPRD until 2 years before the diagnosis date (corresponding date in controls) and in the 5 years before the diagnosis date were separately assessed. Conditional logistic regression analyses were used to generate odds ratios (ORs) and 95% confidence intervals (CIs) associated with PPI or H₂RA use compared with nonuse.

Results: Ever use of PPIs since entry into the GPRD (excluding the 2 years prior to diagnosis) was not associated with risk of pancreatic cancer; OR (95% CI) 1.02 (0.85 – 1.22). Neither the dose nor the duration of PPI or H₂RA use was associated with pancreatic cancer risk. No consistent patterns of association were seen when cumulative exposure (dose and duration) to these drugs was examined separately or together.

Conclusion: PPI/H₂RA use, in a UK population, was not associated with pancreatic cancer risk.

Sustained Release of the CCR5 Inhibitors CMPD167 and Maraviroc from Vaginal Rings in Rhesus Macaques

Antiretroviral entry inhibitors are now being considered as vaginally administered microbicide candidates for prevention of sexual transmission of human immunodeficiency virus. Previous studies testing the entry inhibitors maraviroc and CMPD167 in aqueous gel formulations showed efficacy in the macaque challenge model, although protection was highly dependent on the time period between initial gel application and subsequent challenge. In this paper, we describe the sustained release of the entry inhibitors maraviroc and CMPD167 from matrix-type silicone elastomer vaginal rings both in vitro and in vivo. Both inhibitors were released continuously over 28 days from rings in vitro, at rates of 100-2500 µg/day. In 28-day pharmacokinetic studies in rhesus macaques, the compounds were measured in the vaginal fluid and vaginal tissue; steady state fluid concentrations were ~106 fold greater than IC50 values for SHIV-162P3 inhibition in macaque lymphocytes in vitro. Plasma concentrations for both compounds were very low. Pretreatment of macaques with Depo-Provera® (DP), as commonly used in macaque challenge studies, was shown to significantly modify the bio-distribution of the inhibitors, but not the overall amount released. Vaginal fluid and tissue concentrations were significantly decreased while plasma levels increased with DP pretreatment. These observations have implications for designing macaque challenge experiments, and also for ring performance during the human female menstrual cycle. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Metallo-aminopeptidase inhibitors

Aminopeptidases are enzymes that selectively hydrolyze an amino acid residue from the N-terminus of proteins and peptides. They are important for the proper functioning of prokaryotic and eukaryotic cells, but very often are central players in the devastating human diseases like cancer, malaria and diabetes. The largest aminopeptidase group include enzymes containing metal ion(s) in their active centers, which often determines the type of inhibitors that are the most suitable for them. Effective ligands mostly bind in a non-covalent mode by forming complexes with the metal ion(s). Here, we present several approaches for the design of inhibitors for metallo-aminopeptidases. The optimized structures should be considered as potential leads in the drug discovery process against endogenous and infectious diseases. Crown Copyright (C) 2010 Published by Elsevier Masson SAS. All rights reserved.

Fluorobenzoyl dipeptidyl derivatives as inhibitors of the Fasciola hepatica cysteine protease cathepsin L1

Cathepsins are known to have many important physiological roles and provide a viable target for inhibition. Fluorobenzoyl dipeptide derivatives were synthesized and tested for biological activity in an effort to find an efficient inhibitor of the cysteine protease cathepsin L. Thirty-six novel inhibitors (1-36) were synthesized from protected amino acids via the standard DCC/HOBt coupling protocol, containing a benzyl ester or a nitrile as an electrophilic warhead. The activity of the inhibitors was evaluated against cathepsin L and IC50 values calculated. Modification of both amino acids and terminal groups afforded compounds with single digit micromolar inhibition. Results utilizing the benzoyl-L-leucine-glycine nitrile backbone are comparable to that for the commercially available inhibitor 39.

Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in cancer progression and survival: a systematic review

Objective: To investigate the association between angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) and disease progression and survival in cancer patients.

Methods: Using terms for cancer and ACEIs/ARBs, MEDLINE, EMBASE and Web of Science were systematically searched for observational/interventional studies that used clinically relevant outcomes for cancer progression and survival.

Results: Ten studies met the inclusion criteria. Two studies showed a significant improvement in overall survival (OS) with ACEI/ARB use among patients with advanced pancreatic (HR 0.52, 95% CI 0.29–0.88) and non-small cell lung cancer (HR 0.56, 95% CI 0.33–0.95). An improvement in progression-free survival (PFS) was also reported for pancreatic cancer patients (HR 0.58, 95% CI 0.34–0.95) and patients with renal cell carcinoma (HR 0.54, p = 0.02). ACEI/ARB use was protective against breast cancer recurrence (HR 0.60, 95% CI 0.37–0.96), colorectal cancer distant metastasis (OR 0.22, 95% CI 0.08–0.65) and prostate specific antigen (PSA) failure in prostate cancer patients (p = 0.034). One study observed a worse OS (HR 2.01, 95% CI 1.00–4.05) and PFS in ACEI users with multiple myeloma (p = 0.085) while another reported an increased risk of breast cancer recurrence (HR = 1.56, 95% CI 1.02–2.39).

Conclusion: There is some evidence to suggest that ACEI or ARB use may be associated with improved outcomes in cancer patients. Larger, more robust studies are required to explore this relationship further.

Design of potent GlyT1 inhibitors: In vitro and in vivo profiles

Inhibitors of Gly transporter type-1 (GlyT1) for the treatment of schizophrenia have been pursued on the basis of the NMDA receptor (R) hypofunction hypothesis, which stems largely from the observation that NMDAR antagonists induce symptoms that more closely mimic those characteristic of schizophrenia than do other classes of psychotic agents. GlyT1 is responsible for uptake of synaptic Gly, an NMDAR co-agonist amino acid, in neuronal populations throughout the forebrain. GlyT1 inhibition thereby potentiates NMDAR activity by increasing synaptic Gly levels. Correspondingly, a large body of data suggests that GlyT1 inhibitors likely confer more comprehensive symptom alleviation than current antipsychotics. To date, a number of small-molecule GlyT1 inhibitors have been reported by the pharmaceutical industry. Developments in the discovery and characterization of GlyT1 inhibitors are discussed in this review.

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROTUBULE INHIBITORS COLCHICINE AND TUBULOZOLE-C ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microtubule inhibitors colchicine (1 x 10(-3) M) and tubulozole-C(1 x 10(-6) M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of Tl secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type 1 cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, 'yolk' globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-speci?c inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as ef?cient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as ‘FLIP inhibitors’ © 2012 Macmillan Publishers Limited.