Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants.

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (ß, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, ß, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and ß Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.

In vitro selection and analysis of SARS-CoV-2 nirmatrelvir resistance mutations contributing to clinical virus resistance surveillance.

To facilitate the detection and management of potential clinical antiviral resistance, in vitro selection of drug-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) against the virus Mpro inhibitor nirmatrelvir (Paxlovid active component) was conducted. Six Mpro mutation patterns containing T304I alone or in combination with T21I, L50F, T135I, S144A, or A173V emerged, with A173V+T304I and T21I+S144A+T304I mutations showing >20-fold resistance each. Biochemical analyses indicated inhibition constant shifts aligned to antiviral results, with S144A and A173V each markedly reducing nirmatrelvir inhibition and Mpro activity. SARS-CoV-2 surveillance revealed that in vitro resistance-associated mutations from our studies and those reported in the literature were rarely detected in the Global Initiative on Sharing All Influenza Data database. In the Paxlovid Evaluation of Protease Inhibition for COVID-19 in High-Risk Patients trial, E166V was the only emergent resistance mutation, observed in three Paxlovid-treated patients, none of whom experienced COVID-19-related hospitalization or death.

A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

Insights into mechanism of glucokinase activation: observation of multiple distinct protein conformations.

Human glucokinase (GK) is a principal regulating sensor of plasma glucose levels. Mutations that inactivate GK are linked to diabetes, and mutations that activate it are associated with hypoglycemia. Unique kinetic properties equip GK for its regulatory role: although it has weak basal affinity for glucose, positive cooperativity in its binding of glucose causes a rapid increase in catalytic activity when plasma glucose concentrations rise above euglycemic levels. In clinical trials, small molecule GK activators (GKAs) have been efficacious in lowering plasma glucose and enhancing glucose-stimulated insulin secretion, but they carry a risk of overly activating GK and causing hypoglycemia. The theoretical models proposed to date attribute the positive cooperativity of GK to the existence of distinct protein conformations that interconvert slowly and exhibit different affinities for glucose. Here we report the respective crystal structures of the catalytic complex of GK and of a GK-glucose complex in a wide open conformation. To assess conformations of GK in solution, we also carried out small angle x-ray scattering experiments. The results showed that glucose dose-dependently converts GK from an apo conformation to an active open conformation. Compared with wild type GK, activating mutants required notably lower concentrations of glucose to be converted to the active open conformation. GKAs decreased the level of glucose required for GK activation, and different compounds demonstrated distinct activation profiles. These results lead us to propose a modified mnemonic model to explain cooperativity in GK. Our findings may offer new approaches for designing GKAs with reduced hypoglycemic risk.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Discovery of Allosteric, Potent, Subtype Selective, and Peripherally Restricted TrkA Kinase Inhibitors.

Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are activated by hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). Moreover, the NGF antibody tanezumab has provided clinical proof of concept for inhibition of the TrkA kinase pathway in pain leading to significant interest in the development of small molecule inhibitors of TrkA. However, achieving TrkA subtype selectivity over TrkB and TrkC via a Type I and Type II inhibitor binding mode has proven challenging and Type III or Type IV allosteric inhibitors may present a more promising selectivity design approach. Furthermore, TrkA inhibitors with minimal brain availability are required to deliver an appropriate safety profile. Herein, we describe the discovery of a highly potent, subtype selective, peripherally restricted, efficacious, and well-tolerated series of allosteric TrkA inhibitors that culminated in the delivery of candidate quality compound 23.

Discovery of Potent, Selective, and Peripherally Restricted Pan-Trk Kinase Inhibitors for the Treatment of Pain.

Hormones of the neurotrophin family, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), are known to activate the family of Tropomyosin receptor kinases (TrkA, TrkB, and TrkC). Moreover, inhibition of the TrkA kinase pathway in pain has been clinically validated by the NGF antibody tanezumab, leading to significant interest in the development of small molecule inhibitors of TrkA. Furthermore, Trk inhibitors having an acceptable safety profile will require minimal brain availability. Herein, we discuss the discovery of two potent, selective, peripherally restricted, efficacious, and well-tolerated series of pan-Trk inhibitors which successfully delivered three candidate quality compounds 10b, 13b, and 19. All three compounds are predicted to possess low metabolic clearance in human that does not proceed via aldehyde oxidase-catalyzed reactions, thus addressing the potential clearance prediction liability associated with our current pan-Trk development candidate PF-06273340.

Structural insights into the avian AICAR transformylase mechanism.

ATIC encompasses both AICAR transformylase and IMP cyclohydrolase activities that are responsible for the catalysis of the penultimate and final steps of the purine de novo synthesis pathway. The formyl transfer reaction catalyzed by the AICAR Tfase domain is substantially more demanding than that catalyzed by the other folate-dependent enzyme of the purine biosynthesis pathway, GAR transformylase. Identification of the AICAR Tfase active site and key catalytic residues is essential to elucidate how the non-nucleophilic AICAR amino group is activated for formyl transfer. Hence, the crystal structure of dimeric avian ATIC was determined as a complex with the AICAR Tfase substrate AICAR, as well as with an IMP cyclohydrolase inhibitor, XMP, to 1.93 A resolution. AICAR is bound at the dimer interface of the transformylase domains and forms an extensive hydrogen bonding network with a multitude of active site residues. The crystal structure suggests that the conformation of the 4-carboxamide of AICAR is poised to increase the nucleophilicity of the C5 amine, while proton abstraction occurs via His(268) concomitant with formyl transfer. Lys(267) is likely to be involved in the stabilization of the anionic formyl transfer transition state and in subsequent protonation of the THF leaving group.

Structural insights into the human and avian IMP cyclohydrolase mechanism via crystal structures with the bound XMP inhibitor.

Within de novo purine biosynthesis, the AICAR transformylase and IMP cyclohydrolase activities of the bifunctional enzyme ATIC convert the intermediate AICAR to the final product of the pathway, IMP. Identification of the AICAR transformylase active site and a proposed formyl transfer mechanism have already resulted from analysis of crystal structures of avian ATIC in complex with substrate and/or inhibitors. Herein, we focus on the IMPCH active site and the cyclohydrolase mechanism through comparison of crystal structures of XMP inhibitor complexes of human ATIC at 1.9 A resolution with the previously determined avian enzyme. This first human ATIC structure was also determined to ascertain whether any subtle structural differences, compared to the homologous avian enzyme, should be taken into account for structure-based inhibitor design. These structural comparisons, as well as comparative analyses with other IMP and XMP binding proteins, have enabled a catalytic mechanism to be formulated. The primary role of the IMPCH active site appears to be to induce a reconfiguration of the substrate FAICAR to a less energetically favorable, but more reactive, conformer. Backbone (Arg64 and Lys66) and side chain interactions (Thr67) in the IMPCH active site reorient the 4-carboxamide from the preferred conformer that binds to the AICAR Tfase active site to one that promotes intramolecular cyclization. Other backbone amides (Ile126 and Gly127) create an oxyanion hole that helps orient the formyl group for nucleophilic attack by the 4-carboxamide amine and then stabilize the anionic intermediate. Several other residues, including Lys66, Tyr104, Asp125, and Lys137', provide substrate specificity and likely enhance the catalytic rate through contributions to acid-base catalysis.

Crystal structures of human GAR Tfase at low and high pH and with substrate beta-GAR.

Glycinamide ribonucleotide transformylase (GAR Tfase) is a key folate-dependent enzyme in the de novo purine biosynthesis pathway and, as such, has been the target for antitumor drug design. Here, we describe the crystal structures of the human GAR Tfase (purN) component of the human trifunctional protein (purD-purM-purN) at various pH values and in complex with its substrate. Human GAR Tfase exhibits pH-dependent enzyme activity with its maximum around pH 7.5-8. Comparison of unliganded human GAR Tfase structures at pH 4.2 and pH 8.5 reveals conformational differences in the substrate binding loop, which at pH 4.2 occupies the binding cleft and prohibits substrate binding, while at pH 8.5 is permissive for substrate binding. The crystal structure of GAR Tfase with its natural substrate, beta-glycinamide ribonucleotide (beta-GAR), at pH 8.5 confirms this conformational isomerism. Surprisingly, several important structural differences are found between human GAR Tfase and previously reported E. coli GAR Tfase structures, which have been used as the primary template for drug design studies. While the E. coli structure gave valuable insights into the active site and formyl transfer mechanism, differences in structure and inhibition between the bacterial and mammalian enzymes suggest that the human GAR Tfase structure is now the appropriate template for the design of anti-cancer agents.

Structure of avian AICAR transformylase with a multisubstrate adduct inhibitor beta-DADF identifies the folate binding site.

The penultimate catalytic step of the purine de novo synthesis pathway is the conversion of aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formyl-AICAR that requires the cofactor N(10)-formyl-tetrahydrofolate as the formyl donor. This reaction is catalyzed by the AICAR transformylase domain of the bifunctional enzyme AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC). Identification of the location of the AICAR transformylase active site was previously elucidated from the crystal structure of the avian ATIC with bound substrate AICAR; however, due to the absence of any bound folate, the folate binding region of the active site could not be identified. Here, we have determined the homodimeric crystal structure of avian ATIC in complex with the ATIC-specific multisubstrate adduct inhibitor beta-DADF to 2.5 A resolution. Beta-DADF encompasses both the AICAR and folate moieties into a single covalently linked entity, thereby allowing for the characterization of the folate binding pocket of the AICAR transformylase active site. Beta-DADF is intimately bound at the dimer interface of the transformylase domains with the majority of AICAR moiety interactions occurring within one subunit, whereas the primary interactions to the folate occur with the opposing subunit. The crystal structure suggests that a buried Lys(267) is transiently protonated during formyl transfer allowing for the stabilization of the oxyanion transition state and subsequent protonation of N10 on the tetrahydrofolate leaving group. Furthermore, the beta-DADF-bound structure provides a more optimal three-dimensional scaffold to improve the design of specific antineoplastic agents.

Crystal structures of human bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase/IMP cyclohydrolase in complex with potent sulfonyl-containing antifolates.

Aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (ATIC) is a bifunctional enzyme with folate-dependent AICAR transformylase and IMP cyclohydrolase activities that catalyzes the last two steps of purine biosynthesis. The AICAR transformylase inhibitors BW1540 and BW2315 are sulfamido-bridged 5,8-dideazafolate analogs with remarkably potent K(i) values of 8 and 6 nm, respectively, compared with most other antifolates. Crystal structures of ATIC at 2.55 and 2.60 A with each inhibitor, in the presence of substrate AICAR, revealed that the sulfonyl groups dominate inhibitor binding and orientation through interaction with the proposed oxyanion hole. These agents then appear to mimic the anionic transition state and now implicate Asn(431') in the reaction mechanism along with previously identified key catalytic residues Lys(266) and His(267). Potent and selective inhibition of the AICAR transformylase active site, compared with other folate-dependent enzymes, should therefore be pursued by further design of sulfonyl-containing antifolates.