Non-Nucleoside Reverse Transcriptase Inhibitors Join Forces with Integrase Inhibitors to Combat HIV.

In the treatment of acquired immune deficiency syndrome (AIDS), the diarylpyrimidine (DAPY) analogs etravirine (ETR) and rilpivirine (RPV) have been widely effective against human immunodeficiency virus (HIV) variants that are resistant to other non-nucleoside reverse transcriptase inhibitors (NNRTIs). With non-inferior or improved efficacy, better safety profiles, and lower doses or pill burdens than other NNRTIs in the clinic, combination therapies including either of these two drugs have led to higher adherence than other NNRTI-containing treatments. In a separate development, HIV integrase strand transfer inhibitors (INSTIs) have shown efficacy in treating AIDS, including raltegravir (RAL), elvitegravir (EVG), cabotegravir (CAB), bictegravir (BIC), and dolutegravir (DTG). Of these, DTG and BIC perform better against a wide range of resistance mutations than other INSTIs. Nevertheless, drug-resistant combinations of mutations have begun to emerge against all DAPYs and INSTIs, attributable in part to non-adherence. New dual therapies that may promote better adherence combine ETR or RPV with an INSTI and have been safer and non-inferior to more traditional triple-drug treatments. Long-acting dual- and triple-therapies combining ETR or RPV with INSTIs are under study and may further improve adherence. Here, highly resistant emergent mutations and efficacy data on these novel treatments are reviewed. Overall, ETR or RPV, in combination with INSTIs, may be treatments of choice as long-term maintenance therapies that optimize efficacy, adherence, and safety.

Structure of a dihydroxycoumarin active-site inhibitor in complex with the RNase H domain of HIV-1 reverse transcriptase and structure-activity analysis of inhibitor analogs.

Human immunodeficiency virus (HIV) encodes four essential enzymes: protease, integrase, reverse transcriptase (RT)-associated DNA polymerase, and RT-associated ribonuclease H (RNase H). Current clinically approved anti-AIDS drugs target all HIV enzymatic activities except RNase H, which has proven to be a very difficult target for HIV drug discovery. Our high-throughput screening activities identified the dihydroxycoumarin compound F3284-8495 as a specific inhibitor of RT RNase H, with low micromolar potency in vitro. Optimization of inhibitory potency can be facilitated by structural information about inhibitor-target binding. Here, we report the crystal structure of F3284-8495 bound to the active site of an isolated RNase H domain of HIV-1 RT at a resolution limit of 1.71Å. From predictions based on this structure, compounds were obtained that showed improved inhibitory activity. Computational analysis suggested structural alterations that could provide additional interactions with RT and thus improve inhibitory potency. These studies established proof of concept that F3284-8495 could be used as a favorable chemical scaffold for development of HIV RNase H inhibitors.

Identification of alternative binding sites for inhibitors of HIV-1 ribonuclease H through comparative analysis of virtual enrichment studies.

The ribonuclease H (RNase H) domain on the p66 monomer of HIV-1 reverse transcriptase enzyme has become a target for inhibition. The active site is one potential binding site, but other RNase H sites can accommodate inhibitors. Using a combination of experimental and computational studies, potential new binding sites and binding modes have been identified. Libraries of compounds were screened with an experimental assay to identify actives without knowledge of the binding site. The compounds were computationally docked at putative binding sites. Based on positive enrichment of natural-product actives relative to the database of compounds, we propose that many inhibitors bind to an alternative, potentially allosteric, site centered on Q507 of p66. For a series of hydrazone compounds, a small amount of positive enrichment was obtained when active compounds were bound by induced-fit docking at the interface between the DNA:RNA substrate and the RNase H domain near residue Q500.

Synthesis, activity, and structural analysis of novel α-hydroxytropolone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease H.

The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one), potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at noncytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogues can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use.

Structure of HIV-1 reverse transcriptase with the inhibitor beta-Thujaplicinol bound at the RNase H active site.

Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, such as RNH, should be effective against all of the current drug-resistant variants. Here, we present 2.80 A and 2.04 A resolution crystal structures of an RNH inhibitor, beta-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. beta-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that beta-thujaplicinol is a slow-binding RNH inhibitor with noncompetitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

The on-off switch in regulated myosins: different triggers but related mechanisms.

In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long alpha-helical "heavy chain" stabilized by a "regulatory" light chain (RLC) and an "essential" light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca(2+) to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca(2+). Our results indicate that loss of Ca(2+) abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.

Structure and function of HIV-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition.

The rapid replication of HIV-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. In the absence of an effective vaccine, drugs are the only useful treatment. Anti-HIV drugs work; so far drug therapy has saved more than three million years of life. Unfortunately, HIV-1 develops resistance to all of the available drugs. Although a number of useful anti-HIV drugs have been approved for use in patients, the problems associated with drug toxicity and the development of resistance means that the search for new drugs is an ongoing process. The three viral enzymes, reverse transcriptase (RT), integrase (IN), and protease (PR) are all good drug targets. Two distinct types of RT inhibitors, both of which block the polymerase activity of RT, have been approved to treat HIV-1 infections, nucleoside analogs (NRTIs) and nonnucleosides (NNRTIs), and there are promising leads for compounds that either block the RNase H activity or block the polymerase in other ways. A better understanding of the structure and function(s) of RT and of the mechanism(s) of inhibition can be used to generate better drugs; in particular, drugs that are effective against the current drug-resistant strains of HIV-1.

Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design.

HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at approximately 2.5-3.0 A resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 A resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 A resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.

Exploring structural variability in X-ray crystallographic models using protein local optimization by torsion-angle sampling.

Modeling structural variability is critical for understanding protein function and for modeling reliable targets for in silico docking experiments. Because of the time-intensive nature of manual X-ray crystallographic refinement, automated refinement methods that thoroughly explore conformational space are essential for the systematic construction of structurally variable models. Using five proteins spanning resolutions of 1.0-2.8 A, it is demonstrated how torsion-angle sampling of backbone and side-chain libraries with filtering against both the chemical energy, using a modern effective potential, and the electron density, coupled with minimization of a reciprocal-space X-ray target function, can generate multiple structurally variable models which fit the X-ray data well. Torsion-angle sampling as implemented in the Protein Local Optimization Program (PLOP) has been used in this work. Models with the lowest R(free) values are obtained when electrostatic and implicit solvation terms are included in the effective potential. HIV-1 protease, calmodulin and SUMO-conjugating enzyme illustrate how variability in the ensemble of structures captures structural variability that is observed across multiple crystal structures and is linked to functional flexibility at hinge regions and binding interfaces. An ensemble-refinement procedure is proposed to differentiate between variability that is a consequence of physical conformational heterogeneity and that which reflects uncertainty in the atomic coordinates.

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.

HIV-1 reverse transcriptase structure with RNase H inhibitor dihydroxy benzoyl naphthyl hydrazone bound at a novel site.

The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 A resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNA-primed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 A away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.

Crystal structures for HIV-1 reverse transcriptase in complexes with three pyridinone derivatives: a new class of non-nucleoside inhibitors effective against a broad range of drug-resistant strains.

In the treatment of AIDS, the efficacy of all drugs, including non-nucleoside inhibitors (NNRTIs) of HIV-1 reverse transcriptase (RT), has been limited by the rapid appearance of drug-resistant viruses. Lys103Asn, Tyr181Cys, and Tyr188Leu are some of the most common RT mutations that cause resistance to NNRTIs in the clinic. We report X-ray crystal structures for RT complexed with three different pyridinone derivatives, R157208, R165481, and R221239, at 2.95, 2.9, and 2.43 A resolution, respectively. All three ligands exhibit nanomolar or subnanomolar inhibitory activity against wild-type RT, but varying activities against drug-resistant mutants. R165481 and R221239 differ from most NNRTIs in that binding does not involve significant contacts with Tyr181. These compounds strongly inhibit wild-type HIV-1 RT and drug-resistant variants, including Tyr181Cys and Lys103Asn RT. These properties result in part from an iodine atom on the pyridinone ring of both inhibitors that interacts with the main-chain carbonyl oxygen of Tyr188. An acrylonitrile substituent on R165481 substantially improves the activity of the compound against wild-type RT (and several mutants) and provides a way to generate novel inhibitors that could interact with conserved elements of HIV-1 RT at the polymerase catalytic site. In R221239, there is a flexible linker to a furan ring that permits interactions with Val106, Phe227, and Pro236. These contacts appear to enhance the inhibitory activity of R221239 against the HIV-1 strains that carry the Val106Ala, Tyr188Leu, and Phe227Cys mutations.

4-Benzyl and 4-benzoyl-3-dimethylaminopyridin-2(1H)-ones: in vitro evaluation of new C-3-amino-substituted and C-5,6-alkyl-substituted analogues against clinically important HIV mutant strains.

In a program to optimize the anti-HIV activity of the 4-benzyl and 4-benzoyl-3-dimethylaminopyridinones 9 and 10, lead compounds in a new class of highly potent non-nucleoside type inhibitors of HIV-1 reverse transcriptase, modification of the alkyl substitutents at the C-5 and C-6 positions on the pyridinone ring and of the substitutents on the C-3 amino group has been studied. Of the 17 new 5/6-modified analogues prepared, compounds 31b and 32b substituted at C-5 by an extended nonpolar chain containing an ether function and a C-6 methyl group and compound 35 bearing a C-5 ethyl/C-6 hydroxymethyl substituent pattern were selected on the basis of their in vitro activity against wild-type HIV and the three principle mutant strains, K103N, Y181C, and Y188L. When tested further, it was shown that these molecules, and in particular compound 35, are globally more active than 9, 10, and efavirenz against an additional eight single [L100I, K101E, V106A, E138K, V179E, G190A/S, and F227C] and four double HIV mutant strains [L100I + K103N, K101E + K103N, K103N + Y181C, and F227L + V106A], which are clinically relevant. Concerning modulation of the N-3 substituent, 36 new analogues were prepared. Of these, the N-methyl-N-(2-methoxyethyl)-substituted compounds 40, 42, and 62, as well as the doubly modified compounds 77a and 77b, were selected from the initial screen and were subsequently shown to be active at sub-micromolar concentrations (IC(50)'s) against all the other mutant strains except K103N + Y181C and F227L + V106A. Two possible, but distinct, modes of binding of these analogues in RT were suggested from molecular modeling studies. The preferred mode of binding for compound 62, corresponding to the predicted "orientation 1", was revealed in the X-ray crystal structure of the compound 62-RT complex.

Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding.

Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-A x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm =45 degrees to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called "front door." In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-A nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.

Crystal structure of scallop Myosin s1 in the pre-power stroke state to 2.6 a resolution: flexibility and function in the head.

We have extended the X-ray structure determination of the complete scallop myosin head in the pre-power stroke state to 2.6 A resolution, allowing an atomic comparison of the three major (weak actin binding) states of various myosins. We can now account for conformational differences observed in crystal structures in the so-called "pliant region" at the motor domain-lever arm junction between scallop and vertebrate smooth muscle myosins. A hinge, which may contribute to the compliance of the myosin crossbridge, has also been identified for the first time within the regulatory light-chain domain of the lever arm. Analysis of temperature factors of key joints of the motor domain, especially the SH1 helix, provides crystallographic evidence for the existence of the "internally uncoupled" state in diverse isoforms. The agreement between structural and solution studies reinforces the view that the unwinding of the SH1 helix is a part of the cross-bridge cycle in many myosins.