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.

Dissecting the molecular determinants of clinical PARP1 inhibitor selectivity for tankyrase1.

Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2 [TNKS1/2]), which is an important regulator of Wnt/ß-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage TNKS1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib, and veliparib for their interaction with PARP1 and TNKS1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for TNKS1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand-binding sites of PARP1 and TNKS1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt/ß-catenin pathway-related cancers, such as colorectal cancers.

Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19.

The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.

Small molecule inhibitors reveal PTK6 kinase is not an oncogenic driver in breast cancers.

Protein tyrosine kinase 6 (PTK6, or BRK) is aberrantly expressed in breast cancers, and emerging as an oncogene that promotes tumor cell proliferation, migration and evasion. Both kinase-dependent and -independent functions of PTK6 in driving tumor growth have been described, therefore targeting PTK6 kinase activity by small molecule inhibitors as a therapeutic approach to treat cancers remains to be validated. In this study, we identified novel, potent and selective PTK6 kinase inhibitors as a means to investigate the role of PTK6 kinase activity in breast tumorigenesis. We report here the crystal structures of apo-PTK6 and inhibitor-bound PTK6 complexes, providing the structural basis for small molecule interaction with PTK6. The kinase inhibitors moderately suppress tumor cell growth in 2D and 3D cell cultures. However, the tumor cell growth inhibition shows neither correlation with the PTK6 kinase activity inhibition, nor the total or activated PTK6 protein levels in tumor cells, suggesting that the tumor cell growth is independent of PTK6 kinase activity. Furthermore, in engineered breast tumor cells overexpressing PTK6, the inhibition of PTK6 kinase activity does not parallel the inhibition of tumor cell growth with a >500-fold shift in compound potencies (IC50 values). Overall, these findings suggest that the kinase activity of PTK6 does not play a significant role in tumorigenesis, thus providing important evidence against PTK6 kinase as a potential therapeutic target for breast cancer treatment.

Optimization of Orally Bioavailable Enhancer of Zeste Homolog 2 (EZH2) Inhibitors Using Ligand and Property-Based Design Strategies: Identification of Development Candidate (R)-5,8-Dichloro-7-(methoxy(oxetan-3-yl)methyl)-2-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3,4-dihydroisoquinolin-1(2H)-one (PF-06821497).

A new series of lactam-derived EZH2 inhibitors was designed via ligand-based and physicochemical-property-based strategies to address metabolic stability and thermodynamic solubility issues associated with previous lead compound 1. The new inhibitors incorporated an sp3 hybridized carbon atom at the 7-position of the lactam moiety present in lead compound 1 as a replacement for a dimethylisoxazole group. This transformation enabled optimization of the physicochemical properties and potency compared to compound 1. Analysis of relationships between calculated log D (clogD) values and in vitro metabolic stability and permeability parameters identified a clogD range that afforded an increased probability of achieving favorable ADME data in a single molecule. Compound 23a exhibited the best overlap of potency and pharmaceutical properties as well as robust tumor growth inhibition in vivo and was therefore advanced as a development candidate (PF-06821497). A crystal structure of 23a in complex with the three-protein PRC2 complex enabled understanding of the key structural features required for optimal binding.

Conserved RNA-binding specificity of polycomb repressive complex 2 is achieved by dispersed amino acid patches in EZH2.

Polycomb repressive complex 2 (PRC2) is a key chromatin modifier responsible for methylation of lysine 27 in histone H3. PRC2 has been shown to interact with thousands of RNA species in vivo, but understanding the physiological function of RNA binding has been hampered by the lack of separation-of-function mutants. Here, we use comprehensive mutagenesis and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify critical residues for RNA interaction in PRC2 core complexes from Homo sapiens and Chaetomium thermophilum, for which crystal structures are known. Preferential binding of G-quadruplex RNA is conserved, surprisingly using different protein elements. Key RNA-binding residues are spread out along the surface of EZH2, with other subunits including EED also contributing, and missense mutations of some of these residues have been found in cancer patients. The unusual nature of this protein-RNA interaction provides a paradigm for other epigenetic modifiers that bind RNA without canonical RNA-binding motifs.

Proteome-wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors.

Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety.

The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors.

Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes.

Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase-inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallography and mass spectrometry confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clinically relevant concentrations, highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications.

Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance.

Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform.

Discovery of 1-{(3R,4R)-3-[({5-Chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (PF-06459988), a Potent, WT Sparing, Irreversible Inhibitor of T790M-Containing EGFR Mutants.

First generation EGFR TKIs (gefitinib, erlotinib) provide significant clinical benefit for NSCLC cancer patients with oncogenic EGFR mutations. Ultimately, these patients' disease progresses, often driven by a second-site mutation in the EGFR kinase domain (T790M). Another liability of the first generation drugs is severe adverse events driven by inhibition of WT EGFR. As such, our goal was to develop a highly potent irreversible inhibitor with the largest selectivity ratio between the drug-resistant double mutants (L858R/T790M, Del/T790M) and WT EGFR. A unique approach to develop covalent inhibitors, optimization of reversible binding affinity, served as a cornerstone of this effort. PF-06459988 was discovered as a novel, third generation irreversible inhibitor, which demonstrates (i) high potency and specificity to the T790M-containing double mutant EGFRs, (ii) minimal intrinsic chemical reactivity of the electrophilic warhead, (iii) greatly reduced proteome reactivity relative to earlier irreversible EGFR inhibitors, and (iv) minimal activity against WT EGFR.

Effect of water solvation on the lipophilicity of isomeric pyrimidine-carboxamides.

Incorporation of nitrogen is a common medicinal chemistry tactic to reduce logD values. Neighboring group participation influences logD, so the results are isomer dependent. The logD and logP differences observed between isomeric pyrimidines 1, 2 and 3 presumably result when the carbonyl or ether lone pairs are in close proximity to a heterocyclic nitrogen lone pair, recruiting water to bridge between the electron rich atoms. Various lipophilicity calculators did not discriminate between 1 (logD=2.6) and 3 (logD=1.0), but solvation energies using Poisson-Boltzmann and 3D-RISM methods rationalize the observed differences in lipophilicity among pyrimidine carboxamide isomers.

SAH derived potent and selective EZH2 inhibitors.

A series of novel enhancer of zeste homolog 2 (EZH2) inhibitors was designed based on the chemical structure of the histone methyltransferase (HMT) inhibitor SAH (S-adenosyl-l-homocysteine). These nucleoside-based EZH2 inhibitors blocked the methylation of nucleosomes at H3K27 in biochemical assays employing both WT PRC2 complex as well as a Y641N mutant PRC2 complex. The most potent compound, 27, displayed IC50's against both complexes of 270 nM and 70 nM, respectively. To our knowledge, compound 27 is the most potent SAH-derived inhibitor of the EZH2 PRC2 complex yet identified. This compound also displayed improved potency, lipophilic efficiency (LipE), and selectivity profile against other lysine methyltransferases compared with SAH.

Contribution of hydrogen bonds to protein stability.

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

Pyrimidone-based series of glucokinase activators with alternative donor-acceptor motif.

Glucokinase activators are a class of experimental agents under investigation as a therapy for Type 2 diabetes mellitus. An X-ray crystal structure of a modestly potent agent revealed the potential to substitute the common heterocyclic amide donor-acceptor motif for a pyridone moiety. We have successfully demonstrated that both pyridone and pyrimidone heterocycles can be used as a potent donor-acceptor substituent. Several sub-micromolar analogs that possess the desired partial activator profile were synthesized and characterized. Unfortunately, the most potent activators suffered from sub-optimal pharmacokinetic properties. Nonetheless, these donor-acceptor motifs may find utility in other glucokinase activator series or beyond.

EGFR: tale of the C-terminal tail.

The carboxy terminal tail of epidermal growth factor receptor (EGFR) plays a critical role in the regulation of the enzyme activity of the kinase. There is a good structural model for the mechanism by which the C-terminal tail proximal to the kinase domain contributes to the negative regulation of the activity. Its conformation in the active state, conversely, has remained elusive due to its dynamic nature. A recently published structure of EGFR kinase domain shows the conformation of the proximal C-terminal tail in the active kinase. Analysis of this conformational state of the C-terminal tail is presented, and some of the mutagenesis data is revisited.

Insights into the aberrant activity of mutant EGFR kinase domain and drug recognition.

The oncogenicity of the L858R mutant form of the epidermal growth factor receptor (EGFR) in non-small-cell lung cancer is thought to be due to the constitutive activation of its kinase domain. The selectivity of the marketed drugs gefitinib and erlotinib for L858R mutant is attributed to their specific recognition of the active kinase and to weaker ATP binding by L858R EGFR. We present crystal structures showing that neither L858R nor the drug-resistant L858R+T790M EGFR kinase domain is in the constitutively active conformation. Additional co-crystal structures show that gefitinib and dacomitinib, an irreversible anilinoquinazoline derivative currently in clinical development, may not be conformation specific for the active state of the enzyme. Structural data further reveal the potential mode of recognition of one of the autophosphorylation sites in the C-terminal tail, Tyr-1016, by the kinase domain. Biochemical and biophysical evidence suggest that the oncogenic mutations impact the conformational dynamics of the enzyme.

Ack1: activation and regulation by allostery.

The non-receptor tyrosine kinase Ack1 belongs to a unique multi-domain protein kinase family, Ack. Ack is the only family of SH3 domain containing kinases to have an SH3 domain following the kinase domain; others have their SH3 domains preceding the kinase domain. Previous reports have suggested that Ack1 does not require phosphorylation for activation and the enzyme activity of the isolated kinase domain is low relative to other kinases. It has been shown to dimerize in the cellular environment, which augments its enzyme activity. The molecular mechanism of activation, however, remains unknown. Here we present structural and biochemical data on Ack1 kinase domain, and kinase domain+SH3 domain that suggest that Ack1 in its monomeric state is autoinhibited, like EGFR and CDK. The activation of the kinase domain may require N-lobe mediated symmetric dimerization, which may be facilitated by the N-terminal SAM domain. Results presented here show that SH3 domain, unlike in Src family tyrosine kinases, does not directly control the activation state of the enzyme. Instead we speculate that the SH3 domain may play a regulatory role by facilitating binding of the MIG6 homologous region to the kinase domain. We postulate that features of Ack1 activation and regulation parallel those of receptor tyrosine kinase EGFR with some interesting differences.

Specific inhibition of Notch1 signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells.

Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly, Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs, with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival.