Design and Synthesis of Novel HIV-1 NNRTIs with Bicyclic Cores and with Improved Physicochemical Properties.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent cornerstones of current regimens for treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, NNRTIs usually suffer from low aqueous solubility and the emergence of resistant viral strains. In the present work, novel bicyclic NNRTIs derived from etravirine (ETV) and rilpivirine (RPV), bearing modified purine, tetrahydropteridine, and pyrimidodiazepine cores, were designed and prepared. Compounds 2, 4, and 6 carrying the acrylonitrile moiety displayed single-digit nanomolar activities against the wild-type (WT) virus (EC50 = 2.5, 2.7, and 3.0 nM, respectively), where the low nanomolar activity was retained against HXB2 (EC50 = 2.2-2.8 nM) and the K103N and Y181C mutated strains (fold change, 1.2-6.7×). Most importantly, compound 2 exhibited significantly improved phosphate-buffered saline solubility (10.4 µM) compared to ETV and RPV (≪1 µM). Additionally, the binding modes of compounds 2, 4, and 6 to the reverse transcriptase were studied by X-ray crystallography.

Discovery of Lanraplenib (GS-9876): A Once-Daily Spleen Tyrosine Kinase Inhibitor for Autoimmune Diseases.

Spleen tyrosine kinase (SYK) is a critical regulator of signaling in a variety of immune cell types such as B-cells, monocytes, and macrophages. Accordingly, there have been numerous efforts to identify compounds that selectively inhibit SYK as a means to treat autoimmune and inflammatory diseases. We previously disclosed GS-9973 (entospletinib) as a selective SYK inhibitor that is under clinical evaluation in hematological malignancies. However, a BID dosing regimen and drug interaction with proton pump inhibitors (PPI) prevented development of entospletinib in inflammatory diseases. Herein, we report the discovery of a second-generation SYK inhibitor, GS-9876 (lanraplenib), which has human pharmacokinetic properties suitable for once-daily administration and is devoid of any interactions with PPI. Lanraplenib is currently under clinical evaluation in multiple autoimmune indications.

Discovery of Potent and Selective MTH1 Inhibitors for Oncology: Enabling Rapid Target (In)Validation.

We describe the discovery of three structurally differentiated potent and selective MTH1 inhibitors and their subsequent use to investigate MTH1 as an oncology target, culminating in target (in)validation. Tetrahydronaphthyridine 5 was rapidly identified as a highly potent MTH1 inhibitor (IC50 = 0.043 nM). Cocrystallization of 5 with MTH1 revealed the ligand in a Φ-cis-N-(pyridin-2-yl)acetamide conformation enabling a key intramolecular hydrogen bond and polar interactions with residues Gly34 and Asp120. Modification of literature compound TH287 with O- and N-linked aryl and alkyl aryl substituents led to the discovery of potent pyrimidine-2,4,6-triamine 25 (IC50 = 0.49 nM). Triazolopyridine 32 emerged as a highly selective lead compound with a suitable in vitro profile and desirable pharmacokinetic properties in rat. Elucidation of the DNA damage response, cell viability, and intracellular concentrations of oxo-NTPs (oxidized nucleoside triphosphates) as a function of MTH1 knockdown and/or small molecule inhibition was studied. Based on our findings, we were unable to provide evidence to further pursue MTH1 as an oncology target.

Elucidating molecular interactions of L-nucleotides with HIV-1 reverse transcriptase and mechanism of M184V-caused drug resistance.

Emtricitabine (FTC) and lamivudine (3TC), containing an oxathiolane ring with unnatural (-)-stereochemistry, are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in anti-HIV therapy. Treatment with FTC or 3TC primarily selects for the HIV-1 RT M184V/I resistance mutations. Here we provide a comprehensive kinetic and structural basis for inhibiting HIV-1 RT by (-)-FTC-TP and (-)-3TC-TP and drug resistance by M184V. (-)-FTC-TP and (-)-3TC-TP have higher binding affinities (1/Kd) for wild-type RT but slower incorporation rates than dCTP. HIV-1 RT ternary crystal structures with (-)-FTC-TP and (-)-3TC-TP corroborate kinetic results demonstrating that their oxathiolane sulfur orients toward the DNA primer 3'-terminus and their triphosphate exists in two different binding conformations. M184V RT displays greater (>200-fold) Kd for the L-nucleotides and moderately higher (>9-fold) Kd for the D-isomers compared to dCTP. The M184V RT structure illustrates how the mutation repositions the oxathiolane of (-)-FTC-TP and shifts its triphosphate into a non-productive conformation.

ASK1 contributes to fibrosis and dysfunction in models of kidney disease.

Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.

Novel (2,6-difluorophenyl)(2-(phenylamino)pyrimidin-4-yl)methanones with restricted conformation as potent non-nucleoside reverse transcriptase inhibitors against HIV-1.

To elucidate the structure-geometry-activity relationship in diarylpyrimidine family (DAPYs) containing carbonyl linker between the central pyrimidine core and phenyl type B-arm, a series of (2,6-difluorophenyl)(2-(phenylamino)pyrimidin-4-yl)methanones was designed, prepared and tested for their anti-HIV-1 activity. The carbonyl linker bearing B phenyl arm was successfully attached at both C-2 and C-4 positions of the central pyrimidine ring using a new synthetic approach. Further modifications of target compounds are present at C-5 position of the pyrimidine ring. In vitro anti-HIV-1 activity study performed on a series of 22 compounds confirmed the crucial importance of both conformational rigidity between phenyl B arm and the pyrimidine core linked through the carbonyl bridge, as well as presence of fluoro substituents in ortho-positions of phenyl B moiety. The most potent derivative of the series, compound 17, having almost perpendicular angle within the two planes made from the B aromatic arm and the pyrimidine ring, exhibited low nanomolar anti-HIV-1 activity (EC50 = 4 nM) with no significant toxicity (CC50 > 57.1 µM).

Discovery of GS-9973, a selective and orally efficacious inhibitor of spleen tyrosine kinase.

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.

Synthesis and biological evaluation of phosphonate analogues of nevirapine.

A series of nevirapine-based analogues containing the phosphonate functionality were prepared and evaluated in vitro against HIV RT. The effect of the phosphonate was evaluated against the wild type and Y181C HIV replication. An in vivo PK study was performed on a select analogue.

New class of HIV-1 integrase (IN) inhibitors with a dual mode of action.

tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.

The HIV-1 reverse transcriptase M184I mutation enhances the E138K-associated resistance to rilpivirine and decreases viral fitness.

BACKGROUND: The registrational phase III clinical trials of the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) rilpivirine (RPV) in combination with two nucleoside/nucleotide RT inhibitors (NRTIs) found a unique genotypic resistance pattern involving the NNRTI mutation E138K with the NRTI mutation M184I. Eighty percent of subjects used emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF); a single tablet regimen of FTC/RPV/TDF is in development. METHODS: HIV-1 with E138K and/or M184V or I mutations were constructed and phenotyped in MT-2 cells and the PhenoSense and Antivirogram assays. Viral fitness was determined using growth competitions. Molecular models of the mutants were constructed from the RT-RPV crystal structure. RESULTS: The E138K mutant showed low-level reduced susceptibility to RPV (2.4-fold), but full susceptibility to FTC and tenofovir (TFV). Viruses with M184V or M184I showed high-level resistance to FTC and full susceptibility to RPV and TFV. Addition of M184I, but not M184V, to E138K, further decreased susceptibility to RPV and maintained FTC resistance. The E138K and M184V or I single and double mutants showed decreased replication fitness compared with wild type. M184V outcompeted M184I when compared directly and in the background of E138K. E138K + M184I was less fit than either E138K or M184I alone. Removing a salt bridge between E138/K101 is implicated in resistance to RPV. CONCLUSIONS: The higher frequency of E138K and M184I among RPV + FTC/TDF virologic failures is due to reduced susceptibility of the single mutants to RPV and FTC and the enhanced resistance to RPV for the double mutant at the cost of decreased viral fitness.

Structural and binding analysis of pyrimidinol carboxylic acid and N-hydroxy quinazolinedione HIV-1 RNase H inhibitors.

HIV-1 RNase H breaks down the intermediate RNA-DNA hybrids during reverse transcription, requiring two divalent metal ions for activity. Pyrimidinol carboxylic acid and N-hydroxy quinazolinedione inhibitors were designed to coordinate the two metal ions in the active site of RNase H. High-resolution (1.4 Å to 2.1 Å) crystal structures were determined with the isolated RNase H domain and reverse transcriptase (RT), which permit accurate assessment of the metal and water environment at the active site. The geometry of the metal coordination suggests that the inhibitors mimic a substrate state prior to phosphodiester catalysis. Surface plasmon resonance studies confirm metal-dependent binding to RNase H and demonstrate that the inhibitors do not bind at the polymerase active site of RT. Additional evaluation of the RNase H site reveals an open protein surface with few additional interactions to optimize active-site inhibitors.

Mechanism of resistance to GS-9148 conferred by the Q151L mutation in HIV-1 reverse transcriptase.

GS-9148 is an investigational phosphonate nucleotide analogue inhibitor of reverse transcriptase (RT) (NtRTI) of human immunodeficiency virus type 1 (HIV-1). This compound is an adenosine derivative with a 2',3'-dihydrofuran ring structure that contains a 2'-fluoro group. The resistance profile of GS-9148 is unique in that the inhibitor can select for the very rare Q151L mutation in HIV-1 RT as a pathway to resistance. Q151L is not stably selected by any of the approved nucleoside or nucleotide analogues; however, it may be a transient intermediate that leads to the related Q151M mutation, which confers resistance to multiple compounds that belong to this class of RT inhibitors. Here, we employed pre-steady-state kinetics to study the impact of Q151L on substrate and inhibitor binding and the catalytic rate of incorporation. Most importantly, we found that the Q151L mutant is unable to incorporate GS-9148 under single-turnover conditions. Interference experiments showed that the presence of GS-9148-diphosphate, i.e., the active form of the inhibitor, does not reduce the efficiency of incorporation for the natural counterpart. We therefore conclude that Q151L severely compromises binding of GS-9148-diphosphate to RT. This effect is highly specific, since we also demonstrate that another NtRTI, tenofovir, is incorporated with selectivity similar to that seen with wild-type RT. Incorporation assays with other related compounds and models based on the RT/DNA/GS-9148-diphosphate crystal structure suggest that the 2'-fluoro group of GS-9148 may cause steric hindrance with the side chain of the Q151L mutant.

Crystal structures of HIV-1 reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278): implications for drug design.

Diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitors (NNRTIs) have inherent flexibility, helping to maintain activity against a wide range of resistance mutations. Crystal structures were determined with wild-type and K103N HIV-1 reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278). These structures reveal a similar binding mode for TMC125 and TMC278, whether bound to wild-type or K103N RT. Comparison to previously published structures reveals differences in binding modes for TMC125 and differences in protein conformation for TMC278.

N1-Alkyl pyrimidinediones as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A series of N1-alkyl pyrimidinediones were designed, synthesized and evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Our efforts identified compound 10b, which represents the lead compound in this series with pharmacokinetics and antiviral potency that may support once-daily dosing.

N1-Heterocyclic pyrimidinediones as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A series of N1-heterocyclic pyrimidinediones were extensively evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Inhibitor 1 is active against NNRTI-resistant viruses including RT mutant K103N. The co-crystal structure of inhibitor 1 with HIV-1 RT revealed that H-bonds are formed with K101 and K103. Efforts to improve the suboptimal pharmacokinetic profile of 1 resulted in the discovery of compound 13, which represents the lead compound in this series with improved pharmacokinetics and similar potency as inhibitor 1.

Visualizing the molecular interactions of a nucleotide analog, GS-9148, with HIV-1 reverse transcriptase-DNA complex.

GS-9148 ([5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl]-phosphonic acid) is a dAMP (2'-deoxyadenosine monophosphate) analog that maintains its antiviral activity against drug-resistant HIV. Crystal structures for HIV-1 reverse transcriptase (RT) bound to double-stranded DNA, ternary complexes with either GS-9148-diphosphate or 2'-deoxyadenosine triphosphate (dATP), and a post-incorporation structure with GS-9148 translocated to the priming site were obtained to gain insight into the mechanism of RT inhibition. The binding of either GS-9148-diphosphate or dATP to the binary RT-DNA complex resulted in the fingers subdomain closing around the incoming substrate. This produced up to a 9 A shift in the tips of the fingers subdomain as it closed toward the palm and thumb subdomains. GS-9148-diphosphate shows a similar binding mode as dATP in the nucleotide-binding site. Residues whose mutations confer resistance to nucleotide/nucleoside RT inhibitors, such as M184, Y115, L74, and K65, show little to no shift in orientation whether GS-9148-diphosphate or dATP is bound. One difference observed in binding is the position of the central ring. The dihydrofuran ring of GS-9148-diphosphate interacts with the aromatic side chain of Y115 more than does the ribose ring of dATP, possibly picking up a favorable pi-pi interaction. The ability of GS-9148-diphosphate to mimic the active-site contacts of dATP may explain its effective inhibition of RT and maintained activity against resistance mutations. Interestingly, the 2'-fluoro moiety of GS-9148-diphosphate was found in close proximity to the Q151 side chain, potentially explaining the observed moderately reduced susceptibly to GS-9148 conferred by Q151M mutation.

RNase H active site inhibitors of human immunodeficiency virus type 1 reverse transcriptase: design, biochemical activity, and structural information.

Pyrimidinol carboxylic acids were designed as inhibitors of HIV-1 RNase H function. These molecules can coordinate to two divalent metal ions in the RNase H active site. Inhibition of enzymatic activity was measured in a biochemical assay, but no antiviral effect was observed. Binding was demonstrated via a solid state structure of the isolated p15-Ec domain of HIV-1 RT showing inhibitor and two Mn(II) ions bound to the RNase H active site.

Mutations in the thumb-connection and RNase H domain of HIV type-1 reverse transcriptase of antiretroviral treatment-experienced patients.

BACKGROUND: Antiretroviral therapy that targets HIV type-1 (HIV-1) reverse transcriptase (RT) can be linked to mutations in the thumb-connection (amino acids [AA] 241-426) and RNase H (AA 427-560) domains, which could affect drug resistance. METHODS: Genotypical and statistical analyses were performed on HIV-1 RT from 100 antiretroviral treatment-naive and 248 antiretroviral treatment-experienced patients, the majority of whom were infected with HIV-1 subtype B. The RT region was analysed in three parts: the polymerase (AA 1-240), thumb-connection (AA 241-426) and RNase H (AA 427-560) domains. RESULTS: The polymerase domain had statistically significant changes between the two groups at 24 AA positions that are known resistance sites. Within the thumb-connection domain, R284 and N348 had statistically significant changes between the groups (P=0.007 and P< or =0.001, respectively). In treatment-experienced patients, 17.3% had R284K, whereas 24.5% had N348I substitutions. Both R284 and N348 were 100% conserved in treatment-naive patients. Within the RNase H domain, only K451 showed a statistically significant change (P

Triazole derivatives as non-nucleoside inhibitors of HIV-1 reverse transcriptase--structure-activity relationships and crystallographic analysis.

A series of 3,4,5-trisubstituted 1,2,4-4H triazole derivatives was synthesized and investigated for HIV-1 reverse transcriptase inhibition. An X-ray structure with HIV-1 RT secured the binding mode and allowed the key interactions with the enzyme to be identified.

Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus.

The thermophilic chemolithotroph, Aquifex aeolicus, expresses a gene product that exhibits both ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase activities. These enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. The domain arrangement in the Aquifex enzyme is reminiscent of the fungal ATP sulfurylase, which contains a C-terminal domain that is homologous to APS kinase yet displays no kinase activity. Rather, in the fungal enzyme, the motif serves as a sulfurylase regulatory domain that binds the allosteric effector 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of true APS kinase. Therefore, the Aquifex enzyme may represent an ancestral homolog of a primitive bifunctional enzyme, from which the fungal ATP sulfurylase may have evolved. In heterotrophic sulfur-assimilating organisms such as fungi, ATP sulfurylase catalyzes the first committed step in sulfate assimilation to produce APS, which is subsequently metabolized to generate all sulfur-containing biomolecules. In contrast, ATP sulfurylase in sulfur chemolithotrophs catalyzes the reverse reaction to produce ATP and sulfate from APS and pyrophosphate. Here, the 2.3 A resolution X-ray crystal structure of Aquifex ATP sulfurylase-APS kinase bifunctional enzyme is presented. The protein dimerizes through its APS kinase domain and contains ADP bound in all four active sites. Comparison of the Aquifex ATP sulfurylase active site with those from sulfate assimilators reveals similar dispositions of the bound nucleotide and nearby residues. This suggests that minor perturbations are responsible for optimizing the kinetic properties for the physiologically relevant direction. The APS kinase active-site lid adopts two distinct conformations, where one conformation is distorted by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase active site. This linkage accounts for the low kinase activity of the enzyme under oxidizing conditions. The thermal stability of the Aquifex enzyme can be explained by the 43% decreased cavity volume found within the protein core.