CDK4/6 inhibitors have revolutionized treatment of hormone receptor positive (HR+), HER2 non-amplified (HER2-) breast cancer. Yet, all "dual" CDK4/6 inhibitors show common dose-limiting hematologic toxicities, foremost neutropenia. This poses challenges to provide these agents at concentrations necessary to extinguish cell cycling in tumors. HR+ breast cancer cells are highly dependent on CDK4 but not CDK6. By contrast, CDK4 is dispensable for human bone marrow derived cells, due to the primary and compensatory role of CDK6 in hematopoiesis. This prompted us to develop atirmociclib (PF-07220060), a next-generation CDK4 selective inhibitor. Atirmociclib's impact on circulating neutrophils was reduced, in proportion with its increase in CDK4 versus CDK6 selectivity. Realized dose intensification led to greater CDK4 inhibition and deeper anti-tumor responses, pointing to CDK4 target coverage as a limiting factor of CDK4/6 inhibitor efficacy. We also highlight combinatorial agents that may counter acquired resistance to CDK4 selective inhibition and widen its clinical application.

Inhibitors of cyclin-dependent kinases 4 and 6 have been shown to be clinically effective for the treatment of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced, or metastatic breast cancer. These agents, however, often show neutropenia, likely due to the role of CDK6 in hematopoiesis. Herein described is the discovery of a series of aminopyrimidine-based selective CDK4 inhibitors. Central to our strategy were efficiency-based optimization (LipE and LipMetE), structure-based drug design, and molecular dynamics simulation. The culmination of these efforts resulted in the discovery of PF-07220060 (atirmociclib), which possessed high potency and levels of selectivity for CDK4 over CDK6 that translated to minimal impact on neutrophils while driving efficacy in a mouse ZR75-1 xenograft model.

Abstract PF-07220060 is a selective inhibitor of CDK4, displaying ~20-fold and ~4-fold increased selectivity for CDK4 versus CDK6 when compared to palbociclib and abemaciclib/ribociclib, respectively. This translates to less PF-07220060 associated neutropenia in beagle dogs and humans when compared to dual CDK4/6 inhibitors. Because neutropenia is the primary culprit necessitating limited and often interrupted clinical dosing of dual CDK4/6 inhibitors, the advantage of PF-07220060 lies in the flexibility to escalate the drug’s exposure in patients and thus realize near-complete target coverage of the CDK4 oncogene in tumors that are dependent on this kinase. Here we select tumor types/indications that show dependency on CDK4 but not CDK6 and includes cells derived from luminal breast cancer and androgen-receptor positive metastatic prostate cancer. Additionally, we investigate certain drugs with which PF-07220060 may be combined to maximize its efficacy in these tumor indications. PF-07220060 sensitizes HR+ HER2- breast cancer to the estrogen inhibitor, fulvestrant and the degrader, ARV-471 (vepdegestrant). PF-07220060 + vepdegestrant showed significantly longer tumor regrowth delay vs monotherapy groups and vs Palbo + vepdegestrant and vs PF-07220060 + fulvestrant*. Similarly, PF-07220060 sensitizes prostate cancer to the androgen receptor antagonist, enzalutamide. In HR+ HER2- breast cancer cells, the senescent cell fraction was markedly increased by co-treatment with PF-07220060 and fulvestrant versus either drug alone, albeit the combination did not trigger tumor cell death. Further addition of the PI3K inhibitor alpelisib was sufficient to enforce tumor shrinkage in vivo. Alternatively, inhibition of CDK4 plus CDK2 also led to tumor shrinkage in xenograft models of HR+, HER2- breast cancer. Finally, our preclinical data indicate that the propensity of CDK6 to compensate for CDK4 inhibition in these tumor types is limited. Compensation by CDK6 was seen in only a fraction of the evaluated in vitro and in vivo models. Even so, in these instances, PF-07220060’s efficacy remained comparable to palbociclib’s when both drugs were used at their therapeutic doses. We conclude that PF-07220060’s anti-tumor efficacy is broadly superior to currently approved dual CDK4/6 inhibitors. Citation Format: Lars Anders, Bernadette Pascual, Britton Boras, Julie Cianfrogna, Scott Garza, Na Li, Jing Tang Yuan, Mark Moen, Nanni Huser, Gary Gallego, Mehran Jalaie, Sacha Ninkovic, Sujin Cho-Schultz, Hong Shen, John Kath, Klaus Dress, Wade Diehl, Sajiv Nair, Rhys Jones, Jennifer Lafontaine, Anwar Murtaza, Aida Sacaan, Sudhakar Chintharlapalli, Todd VanArsdale. Preclinical development of the CDK4 selective inhibitor PF-07220060: Increased CDK4 versus CDK6 inhibition leads to improved anti-tumor efficacy at therapeutic concentrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 595.

Supplementary Data from Antitumor Activity and Pharmacology of a Selective Focal Adhesion Kinase Inhibitor, PF-562,271

The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe–kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine. The linking of salicylaldehydes to a kinase binding scaffold resulted in the development of reversible, lysine-targeted covalent kinase inhibitors with enhanced residence time.

The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is a frequently dysregulated pathway in human cancer, and PI3Kα is one of the most frequently mutated kinases in human cancer. A PI3Kα-selective inhibitor may provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family. Here, we describe our efforts to discover a PI3Kα-selective inhibitor by applying structure-based drug design (SBDD) and computational analysis. A novel series of compounds, exemplified by 2,2-difluoroethyl (3S)-3-{[2'-amino-5-fluoro-2-(morpholin-4-yl)-4,5'-bipyrimidin-6-yl]amino}-3-(hydroxymethyl)pyrrolidine-1-carboxylate (1) (PF-06843195), with high PI3Kα potency and unique PI3K isoform and mTOR selectivity were discovered. We describe here the details of the design and synthesis program that lead to the discovery of 1.

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.

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.

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 …

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.

Sulfonyl fluoride (SF)-based activity probes have become important tools in chemical biology. Herein, exploiting the relative chemical stability of SF to carry out a number of unprecedented SF-sparing functional group manipulations, we report the chemoselective synthesis of a toolbox of highly functionalized aryl SF monomers that we used to quickly prepare SF chemical biology probes. In addition to SF, the monomers bear an embedded click handle (a terminal alkyne that can perform copper(I)-mediated azide-alkyne cycloaddition). The monomers can be used either as fragments to prepare clickable SF analogues of drugs (biologically active compounds) bearing an aryl ring or, alternatively, attached to drugs as minimalist clickable aryl SF substituents.

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 (0 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

Activating mutations in EGFR confer constitutive activity providing the oncogenic drive in EGFR-mutant NSCLC. First and 2nd generation EGFR tyrosine kinase inhibitors (TKIs) are effective drugs in this setting, but are constrained by dose-limiting toxicities attributed to inhibition of wild type (WT) EGFR and by drug resistance caused, in the majority of cases, via a T790M secondary mutation in EGFR. We report the pharmacology of a novel irreversible 3rd generation EGFR TKI active against EGFR with activating and T790M mutations, but sparing WT EGFR. Our novel 3rd generation EGFR TKI was studied in a variety of in vitro and in vivo models to determine its inhibitory potencies on different EGFR variants, pharmacokinetics (PK), antitumor efficacy, exposure-response relationships, mechanism of action, and predicted human efficacious dose. In enzyme and cell assays, our compound is a highly potent inhibitor of EGFR double mutants (L858R/T790M and Del/T790M) and EGFR activating mutants (L858R and Del), but a weak inhibitor of WT EGFR (26-fold margin over mutant target potencies). Effects on downstream signaling and function indicate the underlying mechanism of the compound is direct inhibition of EGFR, with subsequent inhibition of downstream signaling that results in apoptosis and viable cell decline. In xenograft mouse models, the compound demonstrates tumor growth inhibition and regression at well-tolerated doses in models driven by EGFR double mutants and EGFR activating mutants. The antitumor efficacy is dose-dependent and strongly correlates with inhibition of EGFR phosphorylation and EGFR-mediated downstream signaling, and induction of apoptosis. Plasma concentrations assumed to be sufficient for efficacy (Ceff) were defined using a mathematical model incorporating the plasma levels of the compound, the associated inhibitory effects on EGFR phosphorylation, and the antitumor efficacy in the double and activating mutant xenograft models. Ceff was in agreement across several models and was used with in vitro human PK properties to calculate required human dose. While our compound possesses a similar profile as other recently disclosed 3rd generation EGFR TKIs, this molecule is distinguished by better potency on the activating mutants and by the widest potency margin on WT EGFR. Given that the target potencies and WT margins of 3rd generation EGFR TKIs have been sufficient for tolerated clinical efficacy in preliminary results, it can be inferred that our compound will have similar promise in the clinic. These results support our compound as a novel EGFR TKI with an inhibitory profile and favorable drug-like properties that suggest utility for treating patients with NSCLC with EGFR activating and resistance mutations. Citation Format: Mike Zientek, Sangita Baxi, Henry Cheng, Valeria Fantin, Jun Li Feng, Allison Given, Zelanna Goldberg, Jie Guo, Michelle Hemkens, John Kath, Jennifer Lafontaine, Gary Li, Pramod Mehta, Brion Murray, Sajiv Nair, Simon Planken, Chad Ray, Yuli Wang, Manli Shi, Anand Sistla, Tod Smeal, Greg Stevens, Wei Tan, Paolo Vicini, Marlena Walls, Liu Yang, Min-Jean Yin, Scott L. Weinrich. Characterization of a novel irreversible third generation EGFR TKI that targets T790M-mediated resistant EGFR-mutant NSCLC while sparing wild type EGFR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2594. doi:10.1158/1538-7445.AM2015-2594