Targeting HIV-1 Reverse Transcriptase Using a Fragment-Based Approach.

Human immunodeficiency virus type I (HIV-1) is a retrovirus that infects cells of the host's immune system leading to acquired immunodeficiency syndrome and potentially death. Although treatments are available to prevent its progression, HIV-1 remains a major burden on health resources worldwide. Continued emergence of drug-resistance mutations drives the need for novel drugs that can inhibit HIV-1 replication through new pathways. The viral protein reverse transcriptase (RT) plays a fundamental role in the HIV-1 replication cycle, and multiple approved medications target this enzyme. In this study, fragment-based drug discovery was used to optimize a previously identified hit fragment (compound B-1), which bound RT at a novel site. Three series of compounds were synthesized and evaluated for their HIV-1 RT binding and inhibition. These series were designed to investigate different vectors around the initial hit in an attempt to improve inhibitory activity against RT. Our results show that the 4-position of the core scaffold is important for binding of the fragment to RT, and a lead compound with a cyclopropyl substitution was selected and further investigated. Requirements for binding to the NNRTI-binding pocket (NNIBP) and a novel adjacent site were investigated, with lead compound 27-a minimal but efficient NNRTI-offering a starting site for the development of novel dual NNIBP-Adjacent site inhibitors.

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crystallographic Fragment Screening of the Catalytic Core Domain.

HIV-1 integrase (IN) is essential for virus replication and represents an important multifunctional therapeutic target. Recently discovered quinoline-based allosteric IN inhibitors (ALLINIs) potently impair HIV-1 replication and are currently in clinical trials. ALLINIs exhibit a multimodal mechanism of action by inducing aberrant IN multimerization during virion morphogenesis and by competing with IN for binding to its cognate cellular cofactor LEDGF/p75 during early steps of HIV-1 infection. However, quinoline-based ALLINIs impose a low genetic barrier for the evolution of resistant phenotypes, which highlights a need for discovery of second-generation inhibitors. Using crystallographic screening of a library of 971 fragments against the HIV-1 IN catalytic core domain (CCD) followed by a fragment expansion approach, we have identified thiophenecarboxylic acid derivatives that bind at the CCD-CCD dimer interface at the principal lens epithelium-derived growth factor (LEDGF)/p75 binding pocket. The most active derivative (5) inhibited LEDGF/p75-dependent HIV-1 IN activity in vitro with an IC50 of 72 µm and impaired HIV-1 infection of T cells at an EC50 of 36 µm The identified lead compound, with a relatively small molecular weight (221 Da), provides an optimal building block for developing a new class of inhibitors. Furthermore, although structurally distinct thiophenecarboxylic acid derivatives target a similar pocket at the IN dimer interface as the quinoline-based ALLINIs, the lead compound, 5, inhibited IN mutants that confer resistance to quinoline-based compounds. Collectively, our findings provide a plausible path for structure-based development of second-generation ALLINIs.

Differential isotopic enrichment to facilitate characterization of asymmetric multimeric proteins using hydrogen/deuterium exchange mass spectrometry.

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein-ligand and protein-protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched ((15)N-labeled) p51 and unlabeled p66. To enable detection of (15)N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% (15)N model. Our results demonstrated that (15)N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but (15)N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure.

Structure of the guanylyltransferase domain of human mRNA capping enzyme.

The enzyme guanylyltransferase (GTase) plays a central role in the three-step catalytic process of adding an (m7)GpppN cap cotranscriptionally to nascent mRNA (pre-mRNAs). The 5'-mRNA capping process is functionally and evolutionarily conserved from unicellular organisms to human. However, the GTases from viruses and yeast have low amino acid sequence identity (∼25%) with GTases from mammals that, in contrast, are highly conserved (∼98%). We have defined by limited proteolysis of human capping enzyme residues 229-567 as comprising the minimum enzymatically active human GTase (hGTase) domain and have determined the structure by X-ray crystallography. Seven related conformational states of hGTase exist in the crystal. The GTP-binding site is evolutionarily and structurally conserved. The positional variations of the oligonucleotide/oligosaccharide binding fold lid domain over the GTP-binding site provide snapshots of the opening and closing of the active site cleft through a swivel motion. The pattern of conserved surface residues in mammals, but not in yeast, supports the finding that the recognition of the capping apparatus by RNA polymerase II and associated transcription factors is highly conserved in mammals, and the mechanism may differ somewhat from that in yeast. The hGTase structure should help in the design of biochemical and molecular biology experiments to explore the proteinprotein and proteinRNA interactions that ensure regulated transcription of genes in humans and other mammals.

Extension into the entrance channel of HIV-1 reverse transcriptase--crystallography and enhanced solubility.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that feature extension into the entrance channel near Glu138. Complexes of the parent anilinylpyrimidine 1 and the morpholinoethoxy analog 2j with HIV-RT have received crystallographic characterization confirming the designs. Measurement of aqueous solubilities of 2j, 2k, the parent triazene 2a, and other NNRTIs demonstrate profound benefits for addition of the morpholinyl substituent.

Phenyl substituted 3-hydroxypyridin-2(1H)-ones: inhibitors of influenza A endonuclease.

Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is recognized as an attractive target for the development of new agents for the treatment of influenza infection. Our earlier study employing small molecule fragment screening using a high-resolution crystal form of pandemic 2009 H1N1 influenza A endonuclease domain (PAN) resulted in the identification of 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating ligand at the active site of the enzyme. In the present study, several phenyl substituted 3-hydroxypyridin-2(1H)-one compounds were synthesized and evaluated for their ability to inhibit the endonuclease activity as measured by a high-throughput fluorescence assay. Two of the more potent compounds in this series, 16 and 18, had IC50 values of 11 and 23nM in the enzymatic assay, respectively. Crystal structures revealed that these compounds had distinct binding modes that chelate the two active site metal ions (M1 and M2) using only two chelating groups. The SAR and the binding mode of these 3-hydroxypyridin-2-ones provide a basis for developing a new class of anti-influenza drugs.

Halo Library, a Tool for Rapid Identification of Ligand Binding Sites on Proteins Using Crystallographic Fragment Screening.

X-ray crystallographic fragment screening (XCFS) uses fragment-sized molecules (∼60 to 300 Da) to access binding sites on proteins that may be inaccessible to larger drug-like molecules (>300 Da). Previous studies have shown that fragments containing halogen atoms bind more often to proteins than non-halogenated fragments. Here, we designed the Halo Library containing 46 halogenated fragments (including the "universal fragment" 4-bromopyrazole), a majority of which have been reported to bind to or inhibit one or more targets. The library was screened against the crystals of HIV-1 reverse transcriptase with the drug rilpivirine, yielding an overall hit rate of 26%. Two new binding sites were discovered, and several hot spots were identified. This small library may thus provide a convenient tool for rapidly assessing the feasibility of a target for XCFS, mapping hot spots and cryptic sites, as well as finding fragment binders that can be useful for developing drug leads.

A comparison of the ability of rilpivirine (TMC278) and selected analogues to inhibit clinically relevant HIV-1 reverse transcriptase mutants.

BACKGROUND: The recently approved anti-AIDS drug rilpivirine (TMC278, Edurant) is a nonnucleoside inhibitor (NNRTI) that binds to reverse transcriptase (RT) and allosterically blocks the chemical step of DNA synthesis. In contrast to earlier NNRTIs, rilpivirine retains potency against well-characterized, clinically relevant RT mutants. Many structural analogues of rilpivirine are described in the patent literature, but detailed analyses of their antiviral activities have not been published. This work addresses the ability of several of these analogues to inhibit the replication of wild-type (WT) and drug-resistant HIV-1. RESULTS: We used a combination of structure activity relationships and X-ray crystallography to examine NNRTIs that are structurally related to rilpivirine to determine their ability to inhibit WT RT and several clinically relevant RT mutants. Several analogues showed broad activity with only modest losses of potency when challenged with drug-resistant viruses. Structural analyses (crystallography or modeling) of several analogues whose potencies were reduced by RT mutations provide insight into why these compounds were less effective. CONCLUSIONS: Subtle variations between compounds can lead to profound differences in their activities and resistance profiles. Compounds with larger substitutions replacing the pyrimidine and benzonitrile groups of rilpivirine, which reorient pocket residues, tend to lose more activity against the mutants we tested. These results provide a deeper understanding of how rilpivirine and related compounds interact with the NNRTI binding pocket and should facilitate development of novel inhibitors.

Fragment screening and HIV therapeutics.

Fragment screening has proven to be a powerful alternative to traditional methods for drug discovery. Biophysical methods, such as X-ray crystallography, NMR spectroscopy, and surface plasmon resonance, are used to screen a diverse library of small molecule compounds. Although compounds identified via this approach have relatively weak affinity, they provide a good platform for lead development and are highly efficient binders with respect to their size. Fragment screening has been utilized for a wide range of targets, including HIV-1 proteins. Here, we review the fragment screening studies targeting HIV-1 proteins using X-ray crystallography or surface plasmon resonance. These studies have successfully detected binding of novel fragments to either previously established or new sites on HIV-1 protease and reverse transcriptase. In addition, fragment screening against HIV-1 reverse transcriptase has been used as a tool to better understand the complex nature of ligand binding to a flexible target.

Cryo-EM structure of the HIV-1 Pol polyprotein provides insights into virion maturation.

Key proteins of retroviruses and other RNA viruses are translated and subsequently processed from polyprotein precursors by the viral protease (PR). Processing of the HIV Gag-Pol polyprotein yields the HIV structural proteins and enzymes. Structures of the mature enzymes PR, reverse transcriptase (RT), and integrase (IN) aided understanding of catalysis and design of antiretrovirals, but knowledge of the Pol precursor architecture and function before PR cleavage is limited. We developed a system to produce stable HIV-1 Pol and determined its cryo-electron microscopy structure. RT in Pol has a similar arrangement to the mature RT heterodimer, and its dimerization may draw together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate assembly and maturation of polyprotein precursors.

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.

Two-dimensional infrared spectra reveal relaxation of the nonnucleoside inhibitor TMC278 complexed with HIV-1 reverse transcriptase.

The two nitrile groups at the wings of the nonnucleoside HIV-1 reverse transcriptase (RT) inhibitor TMC278 are both identified in high-sensitivity 2D IR spectroscopy experiments of the HIV-1 RT/TMC278 complex. The vibrational spectra indicate that the two arms of the inhibitor sense quite different environments within the hydrophobic pocket. The vibrational relaxation of the two arms are almost equal at 3 ps from model studies. The 2D IR spectra expose a significant distribution of nitrile frequencies that diffuse at equilibrium on ultrafast time scales ranging from hundreds of femtoseconds to tens of picoseconds. The slow spectral diffusion of the cyanovinyl arm of the inhibitor is attributed to its interaction with the backbone and side chains in the hydrophobic tunnel. The results show that the inhibitor cyano modes lose memory of their structural configurations relative to the hydrophobic pocket within tens of picoseconds. The cross-peaks between the two arms of the drug are tentatively attributed to relaxation of the nitrile state with both arms excited.

High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: strategic flexibility explains potency against resistance mutations.

TMC278 is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is highly effective in treating wild-type and drug-resistant HIV-1 infections in clinical trials at relatively low doses ( approximately 25-75 mg/day). We have determined the structure of wild-type HIV-1 RT complexed with TMC278 at 1.8 A resolution, using an RT crystal form engineered by systematic RT mutagenesis. This high-resolution structure reveals that the cyanovinyl group of TMC278 is positioned in a hydrophobic tunnel connecting the NNRTI-binding pocket to the nucleic acid-binding cleft. The crystal structures of TMC278 in complexes with the double mutant K103N/Y181C (2.1 A) and L100I/K103N HIV-1 RTs (2.9 A) demonstrated that TMC278 adapts to bind mutant RTs. In the K103N/Y181C RT/TMC278 structure, loss of the aromatic ring interaction caused by the Y181C mutation is counterbalanced by interactions between the cyanovinyl group of TMC278 and the aromatic side chain of Y183, which is facilitated by an approximately 1.5 A shift of the conserved Y(183)MDD motif. In the L100I/K103N RT/TMC278 structure, the binding mode of TMC278 is significantly altered so that the drug conforms to changes in the binding pocket primarily caused by the L100I mutation. The flexible binding pocket acts as a molecular "shrink wrap" that makes a shape complementary to the optimized TMC278 in wild-type and drug-resistant forms of HIV-1 RT. The crystal structures provide a better understanding of how the flexibility of an inhibitor can compensate for drug-resistance mutations.

Crystal Structure of a Retroviral Polyprotein: Prototype Foamy Virus Protease-Reverse Transcriptase (PR-RT).

In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins.

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.

Integrative structural biology studies of HIV-1 reverse transcriptase binding to a high-affinity DNA aptamer.

The high-resolution crystal structure of HIV-1 reverse transcriptase (RT) bound to a 38-mer DNA hairpin aptamer with low pM affinity was previously described. The high-affinity binding aptamer contained 2'-O-methyl modifications and a seven base-pair GC-rich tract and the structure of the RT-aptamer complex revealed specific contacts between RT and the template strand of the aptamer. Similar to all crystal structures of RT bound to nucleic acid template-primers, the aptamer bound RT with a bend in the duplex DNA. To understand the structural basis for the ultra-high-affinity aptamer binding, an integrative structural biology approach was used. Hydrogen-deuterium exchange coupled to liquid chromatography-mass spectrometry (HDX-MS) was used to examine the structural dynamics of RT alone and in the presence of the DNA aptamer. RT was selectively labeled with 15N to unambiguously identify peptides from each subunit. HDX of unliganded RT shows a mostly stable core. The p66 fingers and thumb subdomains, and the RNase H domain are relatively dynamic. HDX indicates that both the aptamer and a scrambled version significantly stabilize regions of RT that are dynamic in the absence of DNA. No substantial differences in RT dynamics are observed between aptamer and scrambled aptamer binding, despite a large difference in binding affinity. Small-angle X-ray scattering and circular dichroism spectroscopy were used to investigate the aptamer conformation in solution and revealed a pre-bent DNA that possesses both A- and B-form helical character. Both the 2'-O-methyl modifications and the GC tract appear to contribute to an energetically favorable conformation for binding to RT that contributes to the aptamer's ultra-high affinity for RT. The X-ray structure of RT with an RNA/DNA version of the aptamer at 2.8 Å resolution revealed a potential role of the hairpin positioning in affinity. Together, the data suggest that both the 2'-O-methyl modifications and the GC tract contribute to an energetically favorable conformation for high-affinity binding to RT.

Structure of HIV-1 reverse transcriptase/d4TTP complex: Novel DNA cross-linking site and pH-dependent conformational changes.

Stavudine (d4T, 2',3'-didehydro-2',3'-dideoxythymidine) was one of the first chain-terminating nucleoside analogs used to treat HIV infection. We present the first structure of the active, triphosphate form of d4T (d4TTP) bound to a catalytic complex of HIV-1 RT/dsDNA template-primer. We also present a new strategy for disulfide (S-S) chemical cross-linking between N6 of a modified adenine at the second overhang base to I63C in the fingers subdomain of RT. The cross-link site is upstream of the duplex-binding region of RT, however, the structure is very similar to published RT structures with cross-linking to Q258C in the thumb, which suggests that cross-linking at either site does not appreciably perturb the RT/DNA structures. RT has a catalytic maximum at pH 7.5. We determined the X-ray structures of the I63C-RT/dsDNA/d4TTP cross-linked complexes at pH 7, 7.5, 8, 8.5, 9, and 9.5. We found small (~0.5 Å), pH-dependent motions of the fingers subdomain that folds in to form the dNTP-binding pocket. We propose that the pH-activity profile of RT relates to this motion of the fingers. Due to side effects of neuropathy and lipodystrophy, use of d4T has been stopped in most countries, however, chemical modification of d4T might lead to the development of a new class of nucleoside analogs targeting RNA and DNA polymerases.

Aryl and Arylalkyl Substituted 3-Hydroxypyridin-2(1H)-ones: Synthesis and Evaluation as Inhibitors of Influenza†A Endonuclease.

Seasonal influenza infections are associated with an estimated 250-500 000 deaths annually. Resistance to the antiviral M2 ion-channel inhibitors has largely invalidated their clinical utility. Resistance to neuraminidase inhibitors has also been observed in several influenza A virus (IAV) strains. These data have prompted research on inhibitors that target the cap-snatching endonuclease activity of the polymerase acidic protein (PA). Baloxavir marboxil (Xofluza®), recently approved for clinical use, inhibits cap-snatching endonuclease. Resistance to Xofluza® has been reported in both in vitro systems and in the clinic. An X-ray crystallographic screening campaign of a fragment library targeting IAV endonuclease identified 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating agent at the active site. We have reported the structure-activity relationships for 3-hydroxypyridin-2(1H)-ones and 3-hydroxyquinolin-2(1H)-ones as endonuclease inhibitors. These studies identified two distinct binding modes associated with inhibition of this enzyme that are influenced by the presence of substituents at the 5- and 6-positions of 3-hydroxypyridin-2(1H)-ones. Herein we report the structure-activity relationships associated with various para-substituted 5-phenyl derivatives of 6-(p-fluorophenyl)-3-hydroxypyridin-2(1H)-ones and the effect of using naphthyl, benzyl, and naphthylmethyl groups as alternatives to the p-fluorophenyl substituent on their activity as endonuclease inhibitors.

Rapid experimental SAD phasing and hot-spot identification with halogenated fragments.

Through X-ray crystallographic fragment screening, 4-bromopyrazole was discovered to be a 'magic bullet' that is capable of binding at many of the ligand 'hot spots' found in HIV-1 reverse transcriptase (RT). The binding locations can be in pockets that are 'hidden' in the unliganded crystal form, allowing rapid identification of these sites for in silico screening. In addition to hot-spot identification, this ubiquitous yet specific binding provides an avenue for X-ray crystallographic phase determination, which can be a significant bottleneck in the determination of the structures of novel proteins. The anomalous signal from 4-bromopyrazole or 4-iodopyrazole was sufficient to determine the structures of three proteins (HIV-1 RT, influenza A endonuclease and proteinase K) by single-wavelength anomalous dispersion (SAD) from single crystals. Both compounds are inexpensive, readily available, safe and very soluble in DMSO or water, allowing efficient soaking into crystals.

Advantages of crystallographic fragment screening: functional and mechanistic insights from a powerful platform for efficient drug discovery.

X-ray crystallography has been an under-appreciated screening tool for fragment-based drug discovery due to the perception of low throughput and technical difficulty. Investigators in industry and academia have overcome these challenges by taking advantage of key factors that contribute to a successful crystallographic screening campaign. Efficient cocktail design and soaking methodologies have evolved to maximize throughput while minimizing false positives/negatives. In addition, technical improvements at synchrotron beamlines have dramatically increased data collection rates thus enabling screening on a timescale comparable to other techniques. The combination of available resources and efficient experimental design has resulted in many successful crystallographic screening campaigns. The three-dimensional crystal structure of the bound fragment complexed to its target, a direct result of the screening effort, enables structure-based drug design while revealing insights regarding protein dynamics and function not readily obtained through other experimental approaches. Furthermore, this "chemical interrogation" of the target protein crystals can lead to the identification of useful reagents for improving diffraction resolution or compound solubility.