Synthesis, biological activity, and ADME properties of novel S-DABOs/N-DABOs as HIV reverse transcriptase inhibitors

Previous studies aimed at exploring the SAR of C2-functionalized S-DABOs demonstrated that the substituent at this position plays a key role in the inhibition of both wild-type RT and drug-resistant enzymes, particularly the K103N mutant form. The introduction of a cyclopropyl group led us to the discovery of a potent inhibitor with picomolar activity against wild-type RT and nanomolar activity against many key mutant forms such as K103N. Despite its excellent antiviral profile, this compound suffers from a suboptimal ADME profile typical of many S-DABO analogues, but it could, however, represent a promising candidate as an anti-HIV microbicide. In the present work, a new series of S-DABO/N-DABO derivatives were synthesized to obtain additional SAR information on the C2-position and in particular to improve ADME properties while maintaining a good activity profile against HIV-1 RT. In vitro ADME properties (PAMPA permeation, water solubility, and metabolic stability) were also experimentally evaluated for the most interesting compounds to obtain a reliable indication of their plasma levels after oral administration.     

Characterization of New DNA Aptamers for Anti-HIV-1 Reverse Transcriptase

HIV-1 RT is a necessary enzyme for retroviral replication, which is the main target for antiviral therapy against AIDS. Effective anti-HIV-1 RT drugs are divided into two groups; nucleoside inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI), which inhibit DNA polymerase. In this study, new DNA aptamers were isolated as anti-HIV-1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild-type (WT) HIV-1 RT and gave a KD value of 75.10±0.29 nM and an IC₅₀ value of 84.81±8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103 N/Y181 C double mutant (KY) HIV-1 RT by around 80 %. Furthermore, the ITC results showed that this aptamer has small binding enthalpies with both WT and KY HIV-1 RTs through which the complex might form a hydrophobic interaction or noncovalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV-1 RTs at the connection domain.     

Design,Synthesis, and SAR of Naphthyl‐Substituted Diarylpyrimidines as Non‐Nucleoside Inhibitors of HIV‐1 Reverse Transcriptase

A series of 38 2‐naphthyl‐substituted diarylpyrimidine (DAPY) analogues, characterized by various substitution patterns on the pyrimidine and naphthalene rings, was synthesized in a straightforward fashion by means of parallel synthesis and evaluated as inhibitors of the HIV‐1 wild‐type and double mutant (K103N+Y181C) strains. Most of the compounds displayed strong activity against wild‐type HIV‐1. The most active compound, with a cyano group at position C6 on the naphthalene ring, exhibited activity against wild‐type HIV‐1 with an EC₅₀ value of 0.002 μM and against the double mutant strain with an EC₅₀ value of 0.24 μM ; the selectivity index (SI) against wild‐type is >180 000, the highest SI value among DAPY analogues. The structure–activity relationship (SAR) of the newly synthesized DAPYs is presented herein.     

Design, synthesis, biological evaluation, and molecular modeling studies of TIBO-like cyclic sulfones as non-nucleoside HIV-1 reverse transcriptase inhibitors

TIBO- and TBO-like sulfone derivatives 1 and 2 were designed, synthesized, and tested for their ability to block the replication cycle of HIV-1 in infected cells. The anti-HIV-1 activities of sulfones 3, which were intermediates in the syntheses of 1 and 2, were also evaluated. Surprisingly, the sulfone analogues of TIBO R82913 (compounds 1) were inactive, whereas interesting results were obtained for truncated derivatives 2. Compound 2 w was the most potent among this series in cell-based assays (EC50=0.07 microM, CC50>200 microM, SI>2857). It was twofold less potent than R82913, but more selective. An X-ray crystallographic analysis was carried out to establish the absolute configuration of 2 w and its enantiomer 2 x, which were obtained by semipreparative HPLC of 2 v, one of the most potent racemates. Compounds 1-3 were proven to target HIV-1 RT. In fact, representative derivatives inhibited recombinant HIV-1 RT in vitro at concentrations similar to those active in cell-based assays. 3D QSAR studies and docking simulations were developed on TIBO- and TBO-like sulfone derivatives to rationalize their anti-HIV-1 potencies and to predict the activity of novel untested sulfone derivatives. Predictive 3D QSAR models were obtained with a receptor-based alignment by docking of TIBO- and TBO-like derivatives into the NNBS of RT.     

Stabilization of the autoproteolysis of TNF-alpha converting enzyme (TACE) results in a novel crystal form suitable for structure-based drug design studies

The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.     

Synthesis and anti-HIV activity of aryl-2-[(4-cyanophenyl)amino]-4-pyrimidinone hydrazones as potent non-nucleoside reverse transcriptase inhibitors

A series of novel diarylpyrimidines (DAPYs) with a ketone hydrazone substituent on the methylene linker between the pyrimidine nucleus and the aryl moiety at the C‐4 position were synthesized, and their antiviral activity against human immunodeficiency virus (HIV)‐1 in MT‐4 cells was evaluated. Most compounds of this class exhibited excellent activity against wild‐type HIV‐1, with EC₅₀ values in the range of 1.7–13.2 nM . Of these compounds, 2‐bromophenyl‐2‐[(4‐cyanophenyl)amino]‐4‐pyrimidinone hydrazone ( 9 k ) displayed the most potent anti‐HIV‐1 activity (EC₅₀=1.7±0.6 nM ), with excellent selectivity for infected over uninfected cells (SI=5762). In addition, the 4‐methyl phenyl analogue 9 d (EC₅₀=2.4±0.2 nM , SI=18461) showed broad spectrum HIV inhibitory activity, with EC₅₀ values of 2.4±0.2 nM against wild‐type HIV‐1, 5.3±0.4 μM against HIV‐1 double‐mutated strain RES056 (K103N+Y181C), and 5.5 μM against HIV‐2 ROD strain. Furthermore, structure–activity relationship (SAR) data and molecular modeling results for these compounds are also discussed.     

Regulation of trypsin activity by Cu2+ chelation of the substrate binding site

Lysine 188 of trypsin was replaced with histidine in order to create a metal chelation site in the substrate binding pocket of this protease, built in a metal binding 'switch,' and to be able to modulate its activity at lower pH. The catalytic properties of wild-type and mutant trypsin were measured with tetrapeptide substrates containing a nitroanilide leaving group and whole native protein substrate: beta- casein. The results obtained reveal that K188H mutation does not affect catalytic efficiency, raising only slightly (from 6 to 8) the arginine/lysine preference of the mutant and increasing 1.8- and 1.2- fold the second-order rate constant k(cat)/Km for arginine- and lysine- containing substrates, respectively. Compared with wild-type trypsin, K188H mutant shows, in the absence of Cu2+, a different catalytic activity pattern as a function of pH. The addition of Cu2+ to trypsin K188H induces a 30-100-fold increase in Km, while k(cat) is scarcely decreased. The hydrolytic activity of this mutant can be fully restored by addition of EDTA. In contrast to a chelating active site, a novel mode of metal-dependent inhibition activity of trypsin with copper is presented. As suggested by molecular modelling studies, the substrate binding pocket of the protease is considerably perturbed by vicinal chelation. More generally, this type of transition metal chelate may present wider possibilities of trypsin activity and specificity modulation than the previously accomplished chelation of a histidine moiety from a catalytic triad.