Influence of the RNase H domain of retroviral reverse transcriptases on the metal specificity and substrate selection of their polymerase domains

Reverse transcriptases from HIV-1 and MuLV respectively prefer Mg2+ and Mn2+ for their polymerase activity, with variable fidelity, on both RNA and DNA templates. The function of the RNase H domain with respect to these parameters is not yet understood. To evaluate this function, two chimeric enzymes were constructed by swapping the RNase H domains between HIV-1 RT and MuLV RT. Chimeric HIV-1 RT, having the RNase H domain of MuLV RT, inherited the divalent cation preference characteristic of MuLV RT on the DNA template with no significant change on the RNA template. Chimeric MuLV RT, likewise partially inherited the metal ion preference of HIV-1 RT. Unlike the wild-type MuLV RT, chimeric MuLV RT is able to use both Mn.dNTP and Mg.dNTP on the RNA template with similar efficiency, while a 30-fold higher preference for Mn.dNTP was seen on the DNA template. The metal preferences for the RNase H activity of chimeric HIV-1 RT and chimeric MuLV RT were, respectively, Mn2+ and Mg2+, a property acquired through their swapped RNase H domains. Chimeric HIV-1 RT displayed higher fidelity and discrimination against rNTPs than against dNTPs substrates, a property inherited from MuLV RT. The overall fidelity of the chimeric MuLV RT was decreased in comparison to the parental MuLV RT, suggesting that the RNase H domain profoundly influences the function of the polymerase domain

In vitro antibacterial activity of rhodanine derivatives against pathogenic clinical isolates

Bacterial infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibacterial drugs. Therefore, new bacterial targets and new antimicrobials are unmet medical needs. Rhodanine derivatives have been shown to possess potent antimicrobial activity via a novel mechanism. However, their potential use as antibacterials has not been fully examined. In this study, we determined the spectrum of activity of seven rhodanine derivatives (compounds Rh 1-7) against clinical isolates of Gram-positive and Gram-negative bacterial strains and Candida albicans. We also synthesized and tested three additional compounds, ethyl ester and amide of rhodanine 2 (Rh 8 and Rh 10, respectively) and ethyl ester of rhodanine 3 (Rh 9) to determine the significance of the carboxyl group modification towards antibacterial activity and human serum albumin binding. A broth microdilution assay confirmed Rh 1-7 exhibit bactericidal activity against Gram-positive pathogens. Rh 2 had significant activity against various vancomycin- resistant (MIC90 = 4 μM) and methicillin-resistant (MIC90 = 4 μM) Staphylococcus aureus (VRSA and MRSA), Staphylococcus epidermidis (MIC = 4 μM) and vancomycinresistant Enterococcus (VRE) strains (MIC90 = 8 μM). The rhodanine compounds exhibited potent activity against Bacillus spp., including Bacillus anthracis, with MIC range of 2-8 μM. In addition, they had potent activity against Clostridium difficile. The most potent compound, Rh 2, at 4 and 8 times its MIC, significantly decreased S. epidermidis biofilm mass by more than 35% and 45%, respectively. None of the rhodanine compounds showed antimicrobial activity (MIC \u3e 128 μM) against various 1) Gram-negative pathogens (Acinetobacter baumannii, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, and Salmonella Typhimurium) or 2) strains of Candida albicans (MIC \u3e 64 μM). The MTS assay confirmed that rhodanines were not toxic to mouse murine macrophage (J774.1A) up to 64 μM, human keratinocytes (HaCat) up to 32 μM, and human ileocecal colorectal cell (HRT-18) up to 128 μM. Overall, these data suggest that certain rhodanine compounds may have potential use for the treatment of several multidrug-resistant Gram-positive bacterial infections. © 2016 AbdelKhalek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors

The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B–RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC50 values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors

The Phosphodiesterase-5 Inhibitor Vardenafil Is a Potent Inhibitor of ABCB1/P-Glycoprotein Transporter

One of the major causes of chemotherapy failure in cancer treatment is multidrug resistance (MDR) which is mediated by the ABCB1/P-glycoprotein. Previously, through the use of an extensive screening process, we found that vardenafil, a phosphodiesterase 5 (PDE-5) inhibitor significantly reverses MDR in ABCB1 overexpressing cancer cells, and its efficacy was greater than that of tadalafil, another PDE-5 inhibitor. The present study was designed to determine the reversal mechanisms of vardenafil and tadalafil on ABC transporters-mediated MDR. Vardenafil or tadalafil alone, at concentrations up to 20 µM, had no significant toxic effects on any of the cell lines used in this study, regardless of their membrane transporter status. However, vardenafil when used in combination with anticancer substrates of ABCB1, significantly potentiated their cytotoxicity in ABCB1 overexpressing cells in a concentration-dependent manner, and this effect was greater than that of tadalafil. The sensitivity of the parenteral cell lines to cytotoxic anticancer drugs was not significantly altered by vardenafil. The differential effects of vardenafil and tadalafil appear to be specific for the ABCB1 transporter as both vardenafil and tadalafil had no significant effect on the reversal of drug resistance conferred by ABCC1 (MRP1) and ABCG2 (BCRP) transporters. Vardenafil significantly increased the intracellular accumulation of [3H]-paclitaxel in the ABCB1 overexpressing KB-C2 cells. In addition, vardenafil significantly stimulated the ATPase activity of ABCB1 and inhibited the photolabeling of ABCB1 with [125I]-IAAP. Furthermore, Western blot analysis indicated the incubation of cells with either vardenafil or tadalafil for 72 h did not alter ABCB1 protein expression. Overall, our results suggest that vardenafil reverses ABCB1-mediated MDR by directly blocking the drug efflux function of ABCB1

The “Cyclopropyl Fragment” is a Versatile Player that Frequently Appears in Preclinical/Clinical Drug Molecules

Recently, there has been an increasing use of the cyclopropyl ring in drug development to transition drug candidates from the preclinical to clinical stage. Important features of the cyclopropane ring are, the (1) coplanarity of the three carbon atoms, (2) relatively shorter (1.51 Å) C–C bonds, (3) enhanced π-character of C–C bonds, and (4) C–H bonds are shorter and stronger than those in alkanes. The present review will focus on the contributions that a cyclopropyl ring makes to the properties of drugs containing it. Consequently, the cyclopropyl ring addresses multiple roadblocks that can occur during drug discovery such as (a) enhancing potency, (b) reducing off-target effects, (c) increasing metabolic stability, (d) increasing brain permeability, (e) decreasing plasma clearance, (f) contributing to an entropically more favorable binding to the receptor, (g) conformational restriction of peptides/peptidomimetics to prevent proteolytic hydrolysis, and (h) altering drug p<i>K</i>_a to reduce its P-glycoprotein efflux ratio

Natural-Products-Inspired Use of the <i>gem</i>-Dimethyl Group in Medicinal Chemistry

The <i>gem</i>-dimethyl moiety is a structural feature frequently found in many natural products of clinical interest, including, but not limited to, taxanes, epothilones, statins, retinoids, di-/triterpenes, noviose deoxysugar, and antibiotics derived from β-lactams, macrolides, and aminocoumarins. Inspired by this time-tested moiety, medicinal chemists have widely explored its use in developing bioactive molecules because of the possibility to (1) increase target engagement, potency, and selectivity through van der Waals interactions and entropically favorable restriction to a bioactive conformation, (2) mitigate toxicity, (3) obtain superior DMPK profile, (4) modulate the p<i>K</i>_a of nearby functionality, (5) induce symmetry into a monomethyl substituted chiral center, and (6) apply the Thorpe–Ingold conformational effect in an <i>o</i>-hydroxydihydro­cinnamic acid based prodrug design. The aim of this Perspective is to illustrate how medicinal chemists have elegantly employed the <i>gem</i>-dimethyl group to obtain clinically useful drugs and to provide synthetic methods to install a <i>gem</i>-dimethyl group