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PDF - 98K, Effect of PF-2771 on breast cell line proliferation and CIN index analysis.

Table S1. Statistics for the crystallographic analysis; Table S2. Biochemical and cellular potencies of the second generation CDK-directed drug AG-024322; Table S3. In vitro analysis of binding potency of abemaciclib, dinaciclib, and palbociclib to non-kinase proteins; Table S4. Broad kinase selectivity of selective CDK4/6 drugs; Table S5. Biochemical dose-response follow-up was conducted for a subset of kinases inhibited by the three CDK4/6 drugs using a Km concentration of ATP (Carna Biosciences) (CDK proteins excluded); Table S6. Kinase selectivity toward endogenous human kinases using irreversible ATP analog target engagement assay; Table S7. Biochemical potencies of selective CDK4/6 drugs palbociclib (PD-0332991); Table S8. Human pharmacokinetic properties of CDK-targeted drugs; Table S9. Isothermal titration calorimetry results for CDK-directed drugs binding to CDK6 in the absence of cyclin D.

Detailed description of the methods used in the manuscript

PDF - 68K, Broad kinase selectivity analysis of PF-2771.

PDF - 76K, Cell cycle analysis of PF-2771 treatment of MDA-MB-468 tumor cells.

PDF - 165K, Comparison of the effect of PF-2771 and a Eg5/KSP inhibitor on the microtubule network.

PDF - 238K, Supplementary Materials and Methods

PDF - 219K, PK/PD/Efficacy model analysis.

Breast cancer patients with tumors lacking the three diagnostic markers (ER, PR, and HER2) are classified as triple-negative (primarily basal-like) and have poor prognosis because there is no disease-specific therapy available. To address this unmet medical need, gene expression analyses using more than a thousand breast cancer samples were conducted, which identified elevated centromere protein E (CENP-E) expression in the basal-a molecular subtype relative to other subtypes. CENP-E, a mitotic kinesin component of the spindle assembly checkpoint, is shown to be induced in basal-a tumor cell lines by the mitotic spindle inhibitor drug docetaxel. CENP-E knockdown by inducible shRNA reduces basal-a breast cancer cell viability. A potent, selective CENP-E inhibitor (PF-2771) was used to define the contribution of CENP-E motor function to basal-like breast cancer. Mechanistic evaluation of PF-2771 in basal-a tumor cells links CENP-E–dependent molecular events (e.g., phosphorylation of histone H3 Ser-10; phospho-HH3-Ser10) to functional outcomes (e.g., chromosomal congression defects). Across a diverse panel of breast cell lines, CENP-E inhibition by PF-2771 selectively inhibits proliferation of basal breast cancer cell lines relative to premalignant ones and its response correlates with the degree of chromosomal instability. Pharmacokinetic–pharmacodynamic efficacy analysis in a basal-a xenograft tumor model shows that PF-2771 exposure is well correlated with increased phospho-HH3-Ser10 levels and tumor growth regression. Complete tumor regression is observed in a patient-derived, basal-a breast cancer xenograft tumor model treated with PF-2771. Tumor regression is also observed with PF-2771 in a taxane-resistant basal-a model. Taken together, CENP-E may be an effective therapeutic target for patients with triple-negative/basal-a breast cancer. Mol Cancer Ther; 13(8); 2104–15. ©2014 AACR.

PDf - 114K, Analysis of mouse body weight in the tumor growth models.

Purpose: Axitinib (AG-013736) is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases 1 to 3 that is in clinical development for the treatment of solid tumors. We provide a comprehensive description of its in vitro characteristics and activities, in vivo antiangiogenesis, and antitumor efficacy and translational pharmacology data.Experimental Design: The potency, kinase selectivity, pharmacologic activity, and antitumor efficacy of axitinib were assessed in various nonclinical models.Results: Axitinib inhibits cellular autophosphorylation of VEGF receptors (VEGFR) with picomolar IC50 values. Counterscreening across multiple kinase and protein panels shows it is selective for VEGFRs. Axitinib blocks VEGF-mediated endothelial cell survival, tube formation, and downstream signaling through endothelial nitric oxide synthase, Akt and extracellular signal-regulated kinase. Following twice daily oral administration, axitinib produces consistent and dose-dependent antitumor efficacy that is associated with blocking VEGFR-2 phosphorylation, vascular permeability, angiogenesis, and concomitant induction of tumor cell apoptosis. Axitinib in combination with chemotherapeutic or targeted agents enhances antitumor efficacy in many tumor models compared with single agent alone. Dose scheduling studies in a human pancreatic tumor xenograft model show that simultaneous administration of axitinib and gemcitabine without prolonged dose interruption or truncation of axitinib produces the greatest antitumor efficacy. The efficacious drug concentrations predicted in nonclinical studies are consistent with the range achieved in the clinic. Although axitinib inhibits platelet-derived growth factor receptors and KIT with nanomolar in vitro potencies, based on pharmacokinetic/pharmacodynamic analysis, axitinib acts primarily as a VEGFR tyrosine kinase inhibitor at the current clinical exposure.Conclusions: The selectivity, potency for VEGFRs, and robust nonclinical activity may afford broad opportunities for axitinib to improve cancer therapy.

Supplementary Figures S1-S7 from Nonclinical Antiangiogenesis and Antitumor Activities of Axitinib (AG-013736), an Oral, Potent, and Selective Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases 1, 2, 3

This abstract was withdrawn by the authors. Citation Format: Dann S, Chionis J, Eisele K, Zhang Q, Liu C, Yuan J, Miller N, Murray B, Xu M, Solowiej J, Wei P, Weinrich S, Sutton S, Behenna D, Ninkovic S, Hoffman R, Freeman-Cook K, Jessen B, Huser N, Zhang C, Visswanathan R, Boras B, VanArsdale T, White MA. Withdrawn [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-06.

NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to TRK inhibitors (larotrectinib and entrectinib) but often mutate, limiting the durability of response for NTRK+ patients. Next-generation inhibitors with compact macrocyclic structures (repotrectinib and selitrectinib) were designed to avoid resistance mutations. Head-to-head potency comparisons of TRK inhibitors and molecular characterization of binding interactions are incomplete, obscuring a detailed understanding of how molecular characteristics translate to potency. Larotrectinib, entrectinib, selitrectinib, and repotrectinib were characterized using cellular models of wild-type TRKA/B/C fusions and resistance mutant variants with a subset evaluated in xenograft tumor models. Crystal structures were determined for repotrectinib bound to TRKA (wild-type, solvent-front mutant). TKI-naïve and pretreated case studies are presented. Repotrectinib was the most potent inhibitor of wild-type TRKA/B/C fusions and was more potent than selitrectinib against all tested resistance mutations, underscoring the importance of distinct features of the macrocycle structures. Cocrystal structures of repotrectinib with wild-type TRKA and the TRKAG595R SFM variant elucidated how differences in macrocyclic inhibitor structure, binding orientation, and conformational flexibility affect potency and mutant selectivity. The SFM crystal structure revealed an unexpected intramolecular arginine sidechain interaction. Repotrectinib caused tumor regression in LMNA–NTRK1 xenograft models harboring GKM, SFM, xDFG, and GKM + SFM compound mutations. Durable responses were observed in TKI-naïve and -pretreated patients with NTRK+ cancers treated with repotrectinib (NCT03093116). This comprehensive analysis of first- and second-generation TRK inhibitors informs the clinical utility, structural determinants of inhibitor potency, and design of new generations of macrocyclic inhibitors.

Control of the cell cycle through selective pharmacological inhibition of CDK4/6 has proven beneficial in the treatment of breast cancer. Extending this level of control to additional cell cycle CDK isoforms represents an opportunity to expand to additional tumor types and potentially provide benefits to patients that develop tumors resistant to selective CDK4/6 inhibitors. However, broad-spectrum CDK inhibitors have a long history of failure due to safety concerns. In this approach, we describe the use of structure-based drug design and Free-Wilson analysis to optimize a series of CDK2/4/6 inhibitors. Further, we detail the use of molecular dynamics simulations to provide insights into the basis for selectivity against CDK9. Based on overall potency, selectivity, and ADME profile, PF-06873600 (22) was identified as a candidate for the treatment of cancer and advanced to phase 1 clinical trials.

Since 2011, with the approval of crizotinib and subsequent approval of four additional targeted therapies, anaplastic lymphoma kinase (ALK) inhibitors have become important treatments for a subset of patients with lung cancer. Each generation of ALK inhibitor showed improvements in terms of central nervous system (CNS) penetration and potency against wild-type (WT) ALK, yet a key continued limitation is their susceptibility to resistance from ALK active-site mutations. The solvent front mutation (G1202R) and gatekeeper mutation (L1196M) are major resistance mechanisms to the first two generations of inhibitors while patients treated with the third-generation ALK inhibitor lorlatinib often experience progressive disease with multiple mutations on the same allele (mutations in cis, compound mutations). TPX-0131 is a compact macrocyclic molecule designed to fit within the ATP-binding boundary to inhibit ALK fusion proteins. In cellular assays, TPX-0131 was more potent than all five approved ALK inhibitors against WT ALK and many types of ALK resistance mutations, e.g., G1202R, L1196M, and compound mutations. In biochemical assays, TPX-0131 potently inhibited (IC50

Exon 20 insertion mutations (Ex20ins) are the third most common type of EGFR oncogenic mutation in lung cancer patients. While several small molecules have been approved (mobocertinib) or are in clinical development (e.g. CLN-081, BDTX-189) none have demonstrated meaningful CNS activity and they can be associated with treatment-limiting adverse events, including wild-type (WT) EGFR-mediated toxicities. LNG-451 is a CNS-penetrant, wild-type EGFR-sparing, covalent inhibitor of EGFR Ex20ins. In cell viability assays dependent on five EGFR Ex20ins mutations, LNG-451 was a potent inhibitor (IC50 7 - 78 nM) including the three most prevalent insertions (V769_D770insASV IC50 = 78 nM, D770_N771insSVD IC50 = 53 nM, H773_V774insNPH IC50 = 75 nM). LNG-451 had low potency in WT EGFR dependent cell assays (WT EGFR Ba/F3 IC50=1,960 nM, cell panel IC50 = 1630 nM). LNG-451 was 21-fold selective for its least potent Ex20ins (V769_D770insASV) relative to WT EGFR. LNG-451 potently inhibited cells dependent on EGFR L861Q (IC50 = 6 nM), EGFR G719S (IC50 = 8 nM), and EGFR G719S/T263P (IC50 = 11 nM). LNG-451 is a highly selective kinase inhibitor (1 μM LNG-451, >90% inhibition for 7 of 409 WT kinases) with screening hits having a similarly positioned cysteine as EGFR. In a Ba/F3 cell-derived xenograft model harboring an EGFR V769_D770insASV exon 20 insertion mutation (EGFR V769_D770insASV CDX), 10 and 50 mg/kg LNG-451 QD PO dosing for 15 days was well-tolerated and resulted in tumor growth inhibition (TGI) of 75.7% and 105.4%, respectively. LNG-451 was efficacious and well-tolerated in the EGFR Ex20ins HuPrime® LU0387 H773-V774insNPH patient-derived xenograft tumor model: 10 mg/kg LNG-451 QD, 67% TGI; 50 mg/kg LNG-451 QD, 91% tumor regression. LNG-451 was highly active in the intracranial PC9-luc xenograft mouse model. In a single dose PK/PD study using the EGFR V769_D770insASV CDX, similar LNG-451 concentrations were observed in tumor, large intestine, and skin tissues (e.g. 50 mg/kg LNG-451 3h post dose: tumor, 8387 ng/mL; large intestine tissue, 14483 ng/mL; skin, 4623 ng/mL). LNG-451 potently suppressed EGFR phosphorylation in tumor tissue (e.g. 99%, 3 h post 50 mg/kg dose) but had minimal-to modest suppression of phospho-EGFR in large intestine tissue (e.g. 4.2%, 3h post 50 mg/kg dose) and skin tissue (e.g 1.9%, 3h post 50 mg/kg dose). Taken together, LNG-451 is a wild-type EGFR-sparing, CNS-penetrant EGFR Ex20ins inhibitor that is expected to provide strong anti-tumor efficacy for patients with advanced/metastatic solid cancers harboring oncogenic EGFR exon 20 insertions with reduced WT EGFR-driven toxicities. Citation Format: Brion W. Murray, Anjali Pandey, Bruce Roth, Tracy Saxton, Daniel J. Estes, Himanshu Agrawal, Santosh Vishwakarma, Gurulingappa Hallur, Ishtiyaque Ahmad, Ravi Trivedi, Helen Jenkins, Paul G. Pearson. LNG-451 is a potent, CNS-penetrant, wild-type EGFR sparing inhibitor of EGFR exon 20 insertion mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3332.

Exon 20 insertion mutations (Ex20ins) are the 3rd most common type of EGFR oncogenic mutation in lung cancer patients and are often associated with brain metastases. While several small molecules have been approved (mobocertinib) or are in clinical development (e.g. CLN-081, BDTX-189) none have demonstrated meaningful CNS activity and they can be associated with treatment-limiting adverse events, including wild-type (WT) EGFR-mediated toxicities. LNG-451 is a potent covalent inhibitor of EGFR Ex20ins (IC50 7 - 78 nM) and other EGFR oncogenic mutations (e.g L858R IC50 = 3 nM, exon 19 deletion IC50 = 24 nM) while being selective against WT EGFR (IC50 = 1630 nM). LNG-451 causes tumor regression in both cell line-derived and patient-derived EGFR Ex20ins-dependent xenograft tumor models. In a rat CNS PK model, a single oral dose of 30 mg/kg LNG-451 achieved high exposures in plasma (AUC0 - 8h = 9771 ng•h/mL) and brain (AUC0 - 8h = 10,041 ng•h/g). In a rat steady-state CNS PK model, unbound partition coefficient for LNG-451 was determined to be Kpu,u = 0.66. To evaluate the potential for LNG-451 to treat metastatic brain disease driven by EGFR mutations, an intracranial PC9-luc xenograft mouse model harboring an EGFR exon 19 deletion mutation was used (LNG-451 cellular EGFR Del19 IC50 = 10.6 nM). LNG-451 had dose-dependent exposures in the plasma and brain with both high mean brain exposure (e.g 1074 ng/g with 25 mg/kg LNG-451) and high mean brain to plasma ratios (e.g. 0.451 for 25 mg/kg LNG-451). LNG-451 treatments were well-tolerated and produced significant anti-tumor activities with 99.5% (2.5 mg/kg LNG-451) and 99.9% (25 mg/kg LNG-451) reductions in total bioluminescence signal (BLI) and regression of tumor BLI (2.5 mg/kg LNG-451 28.8% regression; 25 mg/kg LNG-451, 80.3% regression). An ex vivo imaging analysis of the brain and spinal cords from all animals at the end of the study showed a dose-dependent decrease in the bioluminescent signal in both brain and spinal cords from mice treated with LNG-451 relative to the vehicle control animals. Taken together, LNG-451 is a CNS-penetrant, wild-type EGFR-sparing, EGFR Ex20ins inhibitor that is expected to provide strong anti-tumor efficacy for patients with advanced/metastatic solid cancers harboring oncogenic EGFR exon 20 insertions with reduced WT EGFR driven toxicities. Citation Format: Paul G. Pearson, Anjali Pandey, Bruce Roth, Tracy Saxton, Daniel J. Estes, Ravi Trivedi, Himanshu Agrawal, Gurulingappa Hallur, Ishtiyaque Ahmad, Helen Jenkins, Brion W. Murray. LNG-451, a potent inhibitor of EGFR exon 20 insertion mutations with high CNS exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3261.

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.

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 cysteine 3C-like 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.

Summary The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2− breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G1 kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.

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 ...

NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to first-generation TRK inhibitors (larotrectinib, entrectinib) but the emergence of NTRK mutations limits their efficacy. Next-generation TRK inhibitors (repotrectinib, selitrectinib) have compact macrocyclic structures designed to limit the susceptibility to mutations. However, a detailed understanding of mutant potency and precise binding interactions are lacking. TRK inhibitors were evaluated in cellular models expressing TRKA/B/C fusions with wild-type (WT) and resistance mutations: solvent front (SFM), gatekeeper (GKM), activation loop (xDFG), and compound mutations. In cell proliferation assays, differential potencies against wild type TRKA/B/C fusions were observed: larotrectinib (IC50 23.5 - 49.4 nM), entrectinib (IC50 0.3 - 1.3 nM), selitrectinib (IC50 1.8 - 3.9 nM), and repotrectinib (IC50 < 0.2 nM). First generation TRK inhibitors with extended structures had reduced potency against resistance mutations. Larotrectinib had minimal activity (IC50 >600 nM) against all TRK mutations and entrectinib had >400-fold decreased against all mutations except GKM where there was a range of potencies (IC50 < 0.2 - 60.4 nM). Repotrectinib and selitrectinib were less affected by resistance mutations however repotrectinib was approximately 10-fold more potent than selitrectinib against SFM and compound mutations and 100-fold more potent against GKM. For TRKA/B/C xDFG mutations, repotrectinib had moderate potency (IC50 11.8 - 67.6 nM) while selitrectinib was less potent (IC50 124 - 341 nM). Co-crystal structures of repotrectinib with TRKA and TRKA harboring a SFM provide insight into how subtle differences in macrocyclic inhibitor structure and conformational profiles affect potency and mutant selectivity. Analysis of the first protein structure of a kinase harboring a common SFM (TRKA G595R) revealed unexpected intramolecular interactions which provide insight into its prevalence. Repotrectinib, but not selitrectinib, caused tumor regression in LMNA-NTRKA xenograft models harboring GKM, SFM, or GKM+SFM compound mutations. In the clinic, tumor regression was observed with repotrectinib treatment of a larotrectinib-resistant cholangiocarcinoma patient with both LMNA-TRKA GKM and SFM mutations. Repotrectinib has shown responses in patients with TRK driven tumors with or without resistant mutations in the ongoing global registrational TRIDENT-1 study and has been granted 3 fast track designations. Taken together, the current data characterizes TRK inhibitor potency against resistance mutations and highlights structural characteristics of repotrectinib that enable potent inhibition of TRK proteins and evasion of drug resistance mediated by TRK mutations. Citation Format: Alexander Drilon, Dayong Zhai, Evan Rogers, Wei Deng, Xi Chen, Paul Sprengeler, Siegfried H. Reich, Brion W. Murray. Molecular characteristics of repotrectinib that enable potent inhibition of TRK fusion proteins and broad mutant selectivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1119.

Within the tumor microenvironment (TME), CSF1R (colony stimulating factor 1 receptor) signaling plays a pivotal role in recruitment and enrichment of M2 tumor-associated macrophages (TAM) that dampen the anti-tumor immune response and promote tumor progression. Although CSF1R inhibitors have shown promise in the clinic, tumor cells and TAMs can bypass CSF1R inhibition and expand M2 TAMs through other growth factor receptors, such as MET (hepatocyte growth factor receptor) to confer resistance to targeted therapies and immune checkpoint inhibitors. Through autocrine signaling pathways, CSF1R and MET activation in tumors contribute to tumor invasiveness and metastasis. Therefore, inhibitors that target both CSF1R and MET, such as TPX-0022 (a compact macrocyclic inhibitor of MET/SRC/CSF1R), may more effectively suppress CSF1R mediated signaling in tumor cells and the stroma. In the current study, the role of TPX-0022 in the anti-tumor immune response is evaluated in the context of both normal immune cells and immunocompetent syngeneic murine models. TPX-0022 blocks both HGF and CSF1 mediated signaling and proliferation of MET and CSF1R dependent cell lines. In the MC-38 syngeneic model, TPX-0022 treatment repolarized TAMs towards M1 anti-tumorigenic macrophages, increased CD8 T cells, and inhibited tumor growth. In a mixed lymphocyte reaction assay, we demonstrated that pretreatment of macrophages with TPX-0022 enhances macrophage function in driving the activation and proliferation of CD8 T cells. Furthermore, TPX-0022 treatment of anti-C3/CD28 and or SIINFEKL peptide stimulated murine T cells had no deleterious effects on T cell activation and proliferation. Additionally, TPX-0022 enhanced T cell cytotoxic function towards cancer cells in a dose-dependent manner. Detailed analysis of the TME in CT-26 animals treated with TPX-0022 showed a significant reduction in TAMs, rebalancing both immunosuppressive Treg and M2 macrophages towards anti-tumorigenic CTL (CD8+ CD69+ GnzB+) and M1 macrophages. The combination of TPX-0022 and a PD-1 antibody resulted in significant downregulation of pro-tumorigenic cytokines IL-10, IP-10, IL-1β, and HGF, without impacting the anti-tumor cytokine IFN-γ. In the MC-38 model, TPX-0022 demonstrated single-agent activity and a trend to enhanced efficacy in combination with a PD-1 antibody. Taken together, the preclinical data demonstrates that the MET/SRC/CSF1R inhibitor TPX-0022 can reprogram tumor associated macrophages as well as support antigen presentation and activation of T cells within the TME. A TPX-0022 combination with a PD-1 therapy yielded more anti-tumor activity than single agent treatments. Together, the in vitro and in vivo preclinical data provide a mechanistic rationale for the potential utility of TPX-0022 in combination with immune checkpoint inhibitors. Citation Format: Vikas K. Goel, Wei Deng, Dayong zhai, Nathan V. Lee, Ana Parra, Brion W. Murray. TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1444.

Three generations of ALK inhibitors are approved for the treatment of ALK+ NSCLC but their efficacy is often limited by ALK resistance mutations. The solvent front mutation G1202R and gatekeeper mutation L1196M are major resistance mechanisms to the first two generations of inhibitors. Patients treated with second generation inhibitors are reported to progress with multiple mutations on separate alleles (mutations in trans). In contrast, 35 - 48% of patients treated with lorlatinib progress with multiple mutations on the same allele (compound mutations, mutations in cis). TPX-0131 is an ALK inhibitor with a compact macrocyclic structure designed to bind completely within the ATP binding boundary and overcome a spectrum of single and compound ALK resistant mutations. TPX-0131 was profiled against previous generations of ALK inhibitors both in vitro and in vivo. In biochemical assays, TPX-0131 potently inhibits (IC50 100-fold, crizotinib). TPX-0131 potently inhibits EML4-ALK harboring a G1202R solvent front mutation (IC50 = 0.2 nM) which is >100-fold more potent than previous generations of ALK inhibitors. TPX-0131 potently inhibits ALK harboring a gatekeeper mutation (IC50 = 0.5 nM) and is >10-fold more potent than previous generations of ALK inhibitors. TPX-0131 potently inhibits ALK with a L1198F hinge area mutation (IC50 = 0.2 nM) which is 87 - 3000-fold more potent than previous generations of ALK inhibitors. TPX-0131 is the most potent inhibitor against nine EML4-ALK double and triple compound mutations (6 with IC50 < 1 nM, 3 with IC50 1.6 - 14.9 nM). Evaluation of ALK phosphorylation as a pharmacodynamic marker in tumors showed potent ALK inhibition by TPX-0131 that correlated with TPX-0131 exposure. In Ba/F3 cell-derived xenograft tumor models with EML4-ALK mutations, TPX-0131 (2, 5, 10 mg/kg BID) demonstrated robust anti-tumor activity in the G1202R model (64%, 120%, 200% TGI), G1202R/L1198F model (complete regression, all doses), and G1202R/L1196M model (44%, 83% and 200% TGI). In contrast, lorlatinib (5 mg/kg BID) caused 31% TGI in the G1202R/L1198F model and did not have statistically significant TGI in the G1202R/L1196M model. Taken together, TPX-0131 is a next generation ALK inhibitor that has preclinical potency against WT ALK as well as a broad spectrum of acquired resistance mutations, especially compound mutations, which currently lack any effective ALK inhibitor therapy. Citation Format: Brion W. Murray, Dayong Zhai, Wei Deng, Evan Rogers, Xin Zhang, Jane Ung, Vivian Nguyen, Han Zhang, Maria Barrera, Ana Parra, Jessica Cowell, Dong Lee, Herve Aloysius. TPX-0131, a potent inhibitor of wild type ALK and a broad spectrum of both single and compound ALK resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1469.

KRAS is frequently mutated in multiple cancer types. Therapeutic targeting of KRAS has proven challenging until recent success of KRAS-G12C inhibitor AMG510 that demonstrated tumor regression in lung and colon cancer patients with a KRAS-G12C mutation. However, resistance via feedback reactivation or development of a bypass are expected to limit anti-tumor efficacy of KRAS-G12C inhibitors, favoring a combination approach. Adaptive upregulation of the PI3K/AKT signaling pathway has been recognized as a common resistance mechanism that promotes cancer cell survival and may temper efficacy of KRAS-G12C inhibitors. SRC/FAK signaling promotes tumor cell survival by activation of the PI3K/AKT survival pathway supporting concomitantly targeting SRC/FAK as a promising strategy to potentiate KRAS G12C inhibitors. In addition, oncogenic KRAS induces secretion of various cytokines and growth factors leading to modulation of the tumor microenvironment to promote tumor growth. Targeting the cytokine secretion pathway via JAK2/STAT3 modulation may be important to overcome the resistance to direct KRAS inhibition. Therefore, combinations of KRAS inhibitors with agents that target apoptotic processes and feedback reactivation may represent a promising therapeutic approach. Repotrectinib, a next generation ROS1/TRK/ALK inhibitor, also inhibits SRC/FAK/JAK2 at therapeutically relevant concentrations. Here, we present synergistic effects of repotrectinib in combination with a KRAS-G12C inhibitor in KRAS-G12C mutant cancer models via simultaneous inhibition of SRC/FAK/JAK2. Adding repotrectinib to AMG510 improves AMG510 effectiveness on KRAS-G12C mutant cancer cells with notably increased apoptosis. For example, repotrectinib (1 μM) shifted the antiproliferation IC50 of AMG510 from 213 nM to 3 nM in KRAS-G12C mutant H358 cells. Treatment of H358 cells with AMG510 alone (100 nM) or repotrectinib alone (1 µM) resulted in slight increases in cleaved PARP and caspase-3 proteins, whereas larger increases were observed with the combination. Although high levels of phosphorylated ERK, AKT, STAT3 and FAK were detected in H358 cells, AMG510 (100 nM) was only able to partially modulate phospho-ERK (pERK) with no modulation on pAKT, pSTAT3 and pFAK and repotrectinib (1 μM) suppressed pSTAT3 and pFAK with minimum modulation on pERK and pAKT. The combination in H358 cells results in stronger suppression of pERK and pAKT than either single agent alone, which is consistent with the observed increase in activation of apoptosis. Additional combination data in KRAS-G12C mutant cancer models will be presented. Overall, these studies warrant further clinical investigation on the combination of KRAS-G12C inhibitors with repotrectinib in patients with KRAS-G12C mutation for potential response and duration improvement of current investigational KRAS-G12C inhibitors. Citation Format: J. Jean Cui, Dayong Zhai, Wei Deng, Laura Rodon, Nathan Lee, Brion Murray. Repotrectinib increases effectiveness of KRAS-G12C inhibitors in KRAS-G12C mutant cancer models via simultaneous SRC/FAK/JAK2 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1958.

Kirsten Rat Sarcoma Viral Oncogene homolog (KRAS) is the most frequently mutated oncogene in a broad spectrum of human cancers, including ~30% of non-small cell lung cancer (NSCLC), ~45% of colorectal cancer (CRC) and ~75% of pancreatic cancer. The MEK inhibitor trametinib, which targets the primary downstream signaling pathway activated by mutant KRAS, has shown limited activity when used as a single agent in patients with previously treated KRAS-mutant positive NSCLC. Compensatory upregulation of the PI3K/AKT survival signaling and intrinsic or treatment induced epithelial-to-mesenchymal transition (EMT) are among resistance mechanisms of MEK inhibition in KRAS mutant cancers. SRC/FAK signaling modulates the PI3K/AKT survival pathway and is involved in the integrin mediated EMT signaling. In addition, MEK inhibition has been reported to activate the JAK2/STAT3 pathway to evade therapy. Therefore, the combination of a MEK inhibitor with agents that simultaneously inhibit SRC, FAK and JAK2 may represent a promising therapeutic approach to effectively target KRAS mutant cancers. Repotrectinib, a next generation ROS1/TRK/ALK investigational drug, also inhibits SRC/FAK/JAK2 at therapeutically relevant concentrations. Here, we present synergistic effects of repotrectinib in combination with trametinib in KRAS mutant cancer models. Increased anti-proliferative activity was demonstrated by the combination across panels of KRAS mutant NSCLC and CRC cell lines that harbor a spectrum of KRAS mutations. Additionally, the combination of trametinib and repotrectinib in KRAS mutant cell lines caused an enhanced induction of apoptosis in comparison with trametinib or repotrectinib alone. Trametinib induced a rebound in phospho-AKT (pAKT) levels in a subset of KRAS mutant cells which was substantially suppressed by the combination with repotrectinib. In contrast, suppression of the trametinib-induced pAKT rebound was not achieved by combinations of trametinib with dasatinib (SRCi), ruxolitinib (JAK1/2i), or defactinib (FAKi), suggesting that concomitant SRC/FAK/JAK2 inhibition is potentially necessary. Combination of trametinib and repotrectinib in KRAS mutant murine xenograft studies resulted in suppression of SRC/FAK/STAT3/EGFR/ERK phosphorylation and the enhancement of tumor apoptosis. Importantly, the combination of repotrectinib and trametinib demonstrated increased in vivo anti-tumor effect compared to trametinib alone. Our data suggest that the combination of repotrectinib with trametinib may sustainably suppress the mutant KRAS network signaling to achieve more potent and durable anti-tumor efficacy, and that further clinical investigation of the combination treatment of a MEK inhibitor with repotrectinib in patients with KRAS mutant cancers is warranted. Citation Format: Brion Murray, Wei Deng, Dayong Zhai, Laura Rodon, Nathan Lee, J. Jean Cui. Repotrectinib increases effectiveness of MEK inhibitor trametinib in KRAS mutant cancer models via simultaneous SRC/FAK/JAK2 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1957.

3616 Background: RET fusions/mutations drive oncogenesis in lung and thyroid cancers, and several other malignancies. Selective RET inhibitors (selpercatinib/pralsetinib) are active in patients with these cancers; unfortunately, resistance often occurs. On-target resistance includes the acquisition of solvent front mutations (SFMs i.e. RET G810 substitutions). TPX-0046 is a structurally differentiated RET inhibitor that is potent against a range of RET fusions and mutations including SFMs. Methods: The rationally-designed, compact, macrocyclic RET/SRC inhibitor TPX-0046 was characterized in RET-driven in vitro and in vivo tumor models. Results: In enzymatic assays, TPX-0046 showed low nanomolar potency against wild-type RET and 18 RET mutations/fusions. It was potent against SRC and spared VEGFR2/KDR. TPX-0046 inhibited RET phosphorylation (IC50 < 10 nM) in tumor cell lines (LC2/ad, CCDC6-RET; TT, RET C634W) and Ba/F3 engineered RET models (WT, G810R). In cell proliferation assays, TPX-0046 inhibited KIF5B-RET Ba/F3, LC2/ad, and TT cells with IC50 values ~1 nM. Ba/F3 RET engineered cells with SFMs (e.g. G810C/R/S) were potently inhibited by TPX-0046 (mean proliferation IC50 1–17 nM). TPX-0046 demonstrated marked in vivo anti-tumor efficacy in RET-driven cell-derived and patient-derived xenograft tumor models. In a Ba/F3 KIF5B-RET xenograft model, a single dose of 5 mg/kg TPX-0046 inhibited > 80% of RET phosphorylation (corresponding mean free plasma concentration: 51 nM). At 5 mg/kg BID, tumor regression was observed in RET-dependent xenograft models, including those that harbor RET SFMs: TT, CTG-0838 PDX (NSCLC, KIF5B-RET), CR1520 PDX (CRC, NCOA4-RET), Ba/F3 KIF5B-RET, and Ba/F3 KIF5B-RET G810R. Conclusions: TPX-0046 is a unique next-generation RET inhibitor that possesses potent in vitro and in vivo activity against a diverse range of RET alterations, including SFM-mediated resistance. A phase 1/2 trial for RET inhibitor-resistant and naïve RET-driven cancers is on-going (NCT04161391).

Summary: Alternative splicing is well understood to enhance proteome diversity as cells respond to stimuli. However, mechanistic understanding for how the spliceosome processes precursor messenger RNA (mRNA) transcripts to achieve template diversification is incomplete. We use recently developed enzymatic inhibitors of protein arginine methyltransferase 5 (PRMT5) and human naive T lymphocyte activation as a model system to uncover a precise set of mRNA transcripts that require symmetric arginine dimethylation. This methylation-dependent splicing selectivity is associated with a limited set of signaling pathways that are affected when PRMT5 is inhibited. Specifically, we identify a conserved role for symmetric arginine dimethylation in the induction of antiviral type I and type III interferon signaling following T cell receptor and pattern recognition receptor stimulation in human T lymphocytes and undifferentiated human THP-1 monocytes. Altogether, these findings reveal a mechanism by which cells may be enabled to precisely modulate transcript heterogeneity to orchestrate specific functional outcomes. : Metz et al. employ an optimized chemical toolkit to uncover the unexpected precision with which symmetric arginine dimethylation regulates mRNA splicing. They identify an associated requirement for PRMT5-dependent signaling in the production of antimicrobial type I and type III interferons that is conserved across innate and adaptive immune cells. Keywords: PRMT5, PRMT5 chemical toolkit, precursor mRNA splicing, type I and type III interferon signaling, human innate and adaptive immune system

[][1] In recognition of breast cancer awareness, we highlight advances in breast cancer therapies that build on a long history of discovery yet demonstrate an accelerating pace of development. Breast cancer has challenged physicians and researchers for over 5,000 years ([1][2]). The

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 E...

Protein kinase–directed cancer therapies yield impressive initial clinical responses, but the benefits are typically transient. Enhancing the durability of clinical response is dependent upon patient selection, using drugs with more effective pharmacology, anticipating mechanisms of drug resistance, and applying concerted drug combinations. Achieving these tenets requires an understanding of the targeted kinase's role in signaling networks, how the network responds to drug perturbation, and patient-to-patient network variations. Protein kinases create sophisticated, malleable signaling networks with fidelity coded into the processes that regulate their presence and function. Robust and reliable signaling is facilitated through network processes (e.g., feedback regulation, and compensatory signaling). The routine use of kinase-directed therapies and advancements in both genomic analysis and tumor cell biology are illuminating the complexity of tumor network biology and its capacity to respond to perturbations. Drug efficacy is attenuated by alterations of the drug target (e.g., steric interference, compensatory activity, and conformational changes), compensatory signaling (bypass mechanisms and phenotype switching), and engagement of other oncogenic capabilities (polygenic disease). Factors influencing anticancer drug response and resistance are examined to define the behavior of kinases in network signaling, mechanisms of drug resistance, drug combinations necessary for durable clinical responses, and strategies to identify mechanisms of drug resistance. Mol Cancer Ther; 14(9); 1975–84. ©2015 AACR.

This article describes an integrated rate equation for the time course of covalent enzyme inhibition under the conditions where the substrate concentration is significantly lower than the corresponding Michaelis constant, for example, in the Omnia assays of epidermal growth factor receptor (EGFR) kinase. The newly described method is applicable to experimental conditions where the enzyme concentration is significantly lower than the dissociation constant of the initially formed reversible enzyme-inhibitor complex (no "tight binding"). A detailed comparison with the traditionally used rate equation for covalent inhibition is presented. The two methods produce approximately identical values of the first-order inactivation rate constant (kinact). However, the inhibition constant (Ki), and therefore also the second-order inactivation rate constant kinact/Ki, is underestimated by the traditional method by up to an order of magnitude.

The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30-50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR-ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR-ABL1-driven leukaemia. Axitinib potently inhibited BCR-ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.

Tumors use tryptophan-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO-1) to induce an immunosuppressive environment. IDO-1 is induced in response to inflammatory stimuli and promotes immune tolerance through effector T-cell anergy and enhanced Treg function. As such, IDO-1 is a nexus for the induction of a key immunosuppressive mechanism and represents an important immunotherapeutic target in oncology. Starting from HTS hit 5, IDO-1 inhibitor 6 (EOS200271/PF-06840003) has been developed. The structure–activity relationship around 6 is described and rationalized using the X-ray crystal structure of 6 bound to human IDO-1, which shows that 6, differently from most of the IDO-1 inhibitors described so far, does not bind to the heme iron atom and has a novel binding mode. Clinical candidate 6 shows good potency in an IDO-1 human whole blood assay and also shows a very favorable ADME profile leading to favorable predicted human pharmacokinetic properties, including a predicted half-life of 16–19 h.

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.

Reversible cysteine oxidation is an emerging class of protein post-translational modification (PTM) that regulates catalytic activity, modulates conformation, impacts protein-protein interactions, and affects subcellular trafficking of numerous proteins. Redox PTMs encompass a broad array of cysteine oxidation reactions with different half-lives, topographies, and reactivities such as S-glutathionylation and sulfoxidation. Recent studies from our group underscore the lesser known effect of redox protein modifications on drug binding. To date, biological studies to understand mechanistic and functional aspects of redox regulation are technically challenging. A prominent issue is the lack of tools for labeling proteins oxidized to select chemotype/oxidant species in cells. Predictive computational tools and curated databases of oxidized proteins are facilitating structural and functional insights into regulation of the network of oxidized proteins or redox proteome. In this chapter, we discuss analytical platforms for studying protein oxidation, suggest computational tools currently available in the field to determine redox sensitive proteins, and begin to illuminate roles of cysteine redox PTMs in drug pharmacology.

Reversible cysteine oxidation is an emerging class of protein post-translational modification (PTM) that regulates catalytic activity, modulates conformation, impacts protein-protein interactions, and affects subcellular trafficking of numerous proteins. Redox PTMs encompass a broad array of cysteine oxidation reactions with different half-lives, topographies, and reactivities such as S-glutathionylation and sulfoxidation. Recent studies from our group underscore the lesser known effect of redox protein modifications on drug binding. To date, biological studies to understand mechanistic and functional aspects of redox regulation are technically challenging. A prominent issue is the lack of tools for labeling proteins oxidized to select chemotype/oxidant species in cells. Predictive computational tools and curated databases of oxidized proteins are facilitating structural and functional insights into regulation of the network of oxidized proteins or redox proteome. In this chapter, we discuss analytical platforms for studying protein oxidation, suggest computational tools currently available in the field to determine redox sensitive proteins, and begin to illuminate roles of cysteine redox PTMs in drug pharmacology.

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.

Therapeutically targeting aberrant intracellular kinase signaling is attractive from a biological perspective but drug development is often hindered by toxicities and inadequate efficacy. Predicting drug behaviors using cellular and animal models is confounded by redundant kinase activities, a lack of unique substrates, and cell-specific signaling networks. Cyclin-dependent kinase (CDK) drugs exemplify this phenomenon because they are reported to target common processes yet have distinct clinical activities. Tumor cell studies of ATP-competitive CDK drugs (dinaciclib, AG-024322, abemaciclib, palbociclib, ribociclib) indicate similar pharmacology while analyses in untransformed cells illuminates significant differences. To resolve this apparent disconnect, drug behaviors are described at the molecular level. Nonkinase binding studies and kinome interaction analysis (recombinant and endogenous kinases) reveal that proteins outside of the CDK family appear to have little role in dinaciclib/palbociclib/ribociclib pharmacology, may contribute for abemaciclib, and confounds AG-024322 analysis. CDK2 and CDK6 cocrystal structures with the drugs identify the molecular interactions responsible for potency and kinase selectivity. Efficient drug binding to the unique hinge architecture of CDKs enables selectivity toward most of the human kinome. Selectivity between CDK family members is achieved through interactions with nonconserved elements of the ATP-binding pocket. Integrating clinical drug exposures into the analysis predicts that both palbociclib and ribociclib are CDK4/6 inhibitors, abemaciclib inhibits CDK4/6/9, and dinaciclib is a broad-spectrum CDK inhibitor (CDK2/3/4/6/9). Understanding the molecular components of potency and selectivity also facilitates rational design of future generations of kinase-directed drugs. Mol Cancer Ther; 15(10); 2273–81. ©2016 AACR.

First generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (gefitinib and erlotinib) demonstrate excellent clinical efficacy for NSCLC patients carrying EGFR oncogenic mutations (L858R, del exon 19 deletions between amino acids 746 and 750). Invariable, drug resistance occurs with around 60% of it driven by the EGFR-T790M gatekeeper mutation. To counter the T790M-dependent resistance, third generation covalent EGFR inhibitors have been developed with high potency toward T790M containing mutants and selectivity over WT EGFR. This review provides an overview of the third generation drugs currently in clinical trials and also encompasses novel methodologies developed to discover third generation covalent EGFR drugs.

The c-MET receptor tyrosine kinase is an attractive oncology target because of its critical role in human oncogenesis and tumor progression. An oxindole hydrazide hit 6 was identified during a c-ME...

Pin1 is a member of the cis-trans peptidyl-prolyl isomerase family with potential anti-cancer therapeutic value. Here we report structure-based de novo design and optimization of novel Pin1 inhibitors. Without a viable lead from internal screenings, we designed a series of novel Pin1 inhibitors by interrogating and exploring a protein crystal structure of Pin1. The ligand efficiency of the initial concept molecule was optimized with integrated SBDD and parallel chemistry approaches, resulting in a more attractive lead series.

The c-Met receptor tyrosine kinase (RTK) is a key regulator in cancer, in part, through oncogenic mutations. Eight clinically relevant mutants were characterized by biochemical, biophysical, and cellular methods. The c-Met catalytic domain was highly active in the unphosphorylated state (k(cat) = 1.0 s(-1)) and achieved 160-fold enhanced catalytic efficiency (k(cat)/K(m)) upon activation to 425000 s(-1) M(-1). c-Met mutants had 2-10-fold higher basal enzymatic activity (k(cat)) but achieved maximal activities similar to those of wild-type c-Met, except for Y1235D, which underwent a reduction in maximal activity. Small enhancements of basal activity were shown to have profound effects on the acquisition of full enzymatic activity achieved through accelerating rates of autophosphorylation. Biophysical analysis of c-Met mutants revealed minimal melting temperature differences indicating that the mutations did not alter protein stability. A model of RTK activation is proposed to describe how a RTK response may be matched to a biological context through enzymatic properties. Two c-Met clinical candidates from aminopyridine and triazolopyrazine chemical series (PF-02341066 and PF-04217903) were studied. Biochemically, each series produced molecules that are highly selective against a large panel of kinases, with PF-04217903 (1000-fold selective relative to 208 kinases) being more selective than PF-02341066. Although these prototype inhibitors have similar potencies against wild-type c-Met (K(i) = 6-7 nM), significant differences in potency were observed for clinically relevant mutations evaluated in both biochemical and cellular contexts. In particular, PF-02341066 was 180-fold more active against the Y1230C mutant c-Met than PF-04217903. These highly optimized inhibitors indicate that for kinases susceptible to active site mutations, inhibitor design may need to balance overall kinase selectivity with the ability to inhibit multiple mutant forms of the kinase (penetrance).

Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirus that has a cysteine protease (3CLpro) essential to its life cycle. Steady-state and pre-steady-state kinetic methods were used with highly active 3CLpro to characterize the reaction mechanism. We show that 3CLpro has mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin. The kinetic mechanism for 3CLpro-mediated ester hydrolysis, including the individual rate constants, is consistent with a simple double displacement mechanism. The pre-steady-state burst rate was independent of ester substrate concentration indicating a high commitment to catalysis. When homologous peptidic amide and ester substrates were compared, a series of interesting observations emerged. Despite a 2000-fold difference in nonenzymatic reactivity, highly related amide and ester substrates were found to have similar kinetic parameters in both the steady-state and pre-steady-state. Steady-state solvent isotope effect (SIE) studies showed an inverse SIE for the amide but not ester substrates. Evaluation of the SIE in the pre-steady-state revealed normal SIEs for both amide and ester burst rates. Proton inventory (PI) studies on amide peptide hydrolysis were consistent with two proton-transfer reactions in the transition state while the ester data was consistent with a single proton-transfer reaction. Finally, the pH-inactivation profile of 3CLpro with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data are consistent with a 3CLpro mechanism that utilizes an "electrostatic" trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P1, and a general base-catalyzed deacylation reaction.

First generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (gefitinib and erlotinib) demonstrate excellent clinical efficacy for NSCLC patients carrying EGFR oncogenic mutations (L858R, del exon 19 deletions between amino acids 746 and 750). Invariable, drug resistance occurs with around 60% of it driven by the EGFR-T790M gatekeeper mutation. To counter the T790M-dependent resistance, third generation covalent EGFR inhibitors have been developed with high potency toward T790M containing mutants and selectivity over WT EGFR. This review provides an overview of the third generation drugs currently in clinical trials and also encompasses novel methodologies developed to discover third generation covalent EGFR drugs.

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.

Liquid chromatography-mass spectrometry (LC-MS) has been used extensively in determination of the molecular weights of proteins, as well as covalent protein-ligand complexes. We have successfully developed LC-MS method for protein molecular weight measurement using small-bore and capillary LC-MS under acidic and basic conditions. A high pH method was critical in studying complexes that were unstable under acidic conditions. Microgram sensitivity was achieved using both methods. A protocol to study the binding mode of protein-ligand complexes under denaturing conditions was developed. These methods were applied to CP88 (a proprietary cysteine protease) inhibitors and revealed different binding modes of inhibitors to proteins that had similar non-reversible behavior in biochemical activity assays. The method also confirmed that one inhibitor studied binds to CP88 in a reversible covalent manner.