Discovery of PF-07265028, A Selective Small Molecule Inhibitor of Hematopoietic Progenitor Kinase 1 (HPK1) for the Treatment of Cancer.

Hematopoietic progenitor kinase 1 (HPK1/MAP4K1) represents a high interest target for the treatment of cancer through an immune-mediated mechanism. Herein we present highlights of the drug discovery campaign within the lactam/azalactam series of inhibitors that yielded a small molecule (, PF-07265028), which was advanced to a phase 1 clinical trial (NCT05233436). Key components of the discovery effort included optimization of potency through mitigation of ligand strain as guided by the use of cocrystal structures, mitigation of ADME liabilities (plasma instability and fraction metabolism by CYP2D6), and optimization of kinase selectivity, particularly over immune-modulating kinases with high homology to HPK1.

Discovery of Highly Selective Inhibitors of Microtubule-Associated Serine/Threonine Kinase-like (MASTL).

By virtue of its role in cellular proliferation, microtubule-associated serine/threonine kinase-like (MASTL) represents a novel target and a first-in-class (FIC) opportunity to provide a new impactful therapeutic agent to oncology patients. Herein, we describe a hit-to-lead optimization effort that resulted in the delivery of two highly selective MASTL inhibitors. Key strategies leveraged to enable this work included structure-based drug design (SBDD), analysis of lipophilic efficiency (LipE) and novel synthesis.

Design and Synthesis of Functionally Active 5-Amino-6-Aryl Pyrrolopyrimidine Inhibitors of Hematopoietic Progenitor Kinase 1.

Immune activating agents represent a valuable class of therapeutics for the treatment of cancer. An area of active research is expanding the types of these therapeutics that are available to patients via targeting new biological mechanisms. Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of immune signaling and a target of high interest for the treatment of cancer.

LTK fusions: A new target emerges in non-small cell lung cancer.

Identification of targetable fusions as oncogenic drivers in non-small cell lung cancer has transformed its diagnostic and therapeutic paradigm. In a recent article in Nature, Izumi et al. report the discovery of CLIP1-LTK fusion as a novel oncogenic driver in lung cancer, targetable using the ALK tyrosine kinase inhibitor lorlatinib.

Spectrum of Mechanisms of Resistance to Crizotinib and Lorlatinib in Fusion-Positive Lung Cancer.

Purpose: Current standard initial therapy for advanced, ROS proto-oncogene 1, receptor tyrosine kinase fusion ()-positive (ROS1) non-small cell lung cancer (NSCLC) is crizotinib or entrectinib. Lorlatinib, a next-generation anaplastic lymphoma kinase/ROS1 inhibitor, recently demonstrated efficacy in ROS1 NSCLC, including in crizotinib-pretreated patients. However, mechanisms of lorlatinib resistance in ROS1 disease remain poorly understood.

Multiple conformational states of the HPK1 kinase domain in complex with sunitinib reveal the structural changes accompanying HPK1 trans-regulation.

Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.

Reviving B-Factors: Retrospective Normalized B-Factor Analysis of c-ros Oncogene 1 Receptor Tyrosine Kinase and Anaplastic Lymphoma Kinase L1196M with Crizotinib and Lorlatinib.

Structure-based drug design (SBDD) is commonly leveraged in rational drug design. Usually, ligand and binding site atomic coordinates from crystallographic data are exploited to optimize potency and selectivity. In addition to traditional, static views of proteins and ligands, we propose using normalized B-factors to study protein dynamics as a part of the drug optimization process.

Reviving B-Factors: Activating ALK Mutations Increase Protein Dynamics of the Unphosphorylated Kinase.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that can become oncogenic by activating mutations or overexpression. Full kinetic characterization of both phosphorylated and nonphosphorylated wildtype and mutant ALK kinase domain was done. Our structure-based drug design programs directed at ALK allowed us to interrogate whether X-ray crystallography data could be used to support the hypothesis that activation of ALK by mutation occurs due to increased protein dynamics.

Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound Mutations in ALK-Positive Lung Cancer.

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK.

Lipophilic Efficiency as an Important Metric in Drug Design.

Lipophilic efficiency (LipE) is an important metric that has been increasingly applied in drug discovery medicinal chemistry lead optimization programs. In this Perspective, using literature drug discovery examples, we discuss the concept of rigorously applying LipE to guide medicinal chemistry lead optimization toward drug candidates with potential for superior in vivo efficacy and safety, especially when guided by physiochemical property-based optimization (PPBO). Also highlighted are examples of small structural modifications such as addition of single atoms, small functional groups, and cyclization that produce large increases in LipE.

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.

Conformational Studies and Atropisomerism Kinetics of the ALK Clinical Candidate Lorlatinib (PF-06463922) and Desmethyl Congeners.

Lorlatinib (PF-06463922) is an ALK/ROS1 inhibitor and is in clinical trials for the treatment of ALK positive or ROS1 positive NSCLC (i.e. specific subsets of NSCLC).

Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F.

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor.

CNS Anticancer Drug Discovery and Development Conference White Paper.

Following the first CNS Anticancer Drug Discovery and Development Conference, the speakers from the first 4 sessions and organizers of the conference created this White Paper hoping to stimulate more and better CNS anticancer drug discovery and development. The first part of the White Paper reviews, comments, and, in some cases, expands on the 4 session areas critical to new drug development: pharmacological challenges, recent drug approaches, drug targets and discovery, and clinical paths. Following this concise review of the science and clinical aspects of new CNS anticancer drug discovery and development, we discuss, under the rubric "Accelerating Drug Discovery and Development for Brain Tumors," further reasons why the pharmaceutical industry and academia have failed to develop new anticancer drugs for CNS malignancies and what it will take to change the current status quo and develop the drugs so desperately needed by our patients with malignant CNS tumors.

PF-06463922, an ALK/ROS1 Inhibitor, Overcomes Resistance to First and Second Generation ALK Inhibitors in Preclinical Models.

We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner.

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.

PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations.

Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase.

Discovery of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with preclinical brain exposure and broad-spectrum potency against ALK-resistant mutations.

Although crizotinib demonstrates robust efficacy in anaplastic lymphoma kinase (ALK)-positive non-small-cell lung carcinoma patients, progression during treatment eventually develops. Resistant patient samples revealed a variety of point mutations in the kinase domain of ALK, including the L1196M gatekeeper mutation. In addition, some patients progress due to cancer metastasis in the brain.

Design of potent and selective inhibitors to overcome clinical anaplastic lymphoma kinase mutations resistant to crizotinib.

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients.

Discovery of the Highly Potent PI3K/mTOR Dual Inhibitor PF-04979064 through Structure-Based Drug Design.

PI3K, AKT, and mTOR are key kinases from PI3K signaling pathway being extensively pursued to treat a variety of cancers in oncology. To search for a structurally differentiated back-up candidate to PF-04691502, which is currently in phase I/II clinical trials for treating solid tumors, a lead optimization effort was carried out with a tricyclic imidazo[1,5]naphthyridine series. Integration of structure-based drug design and physical properties-based optimization yielded a potent and selective PI3K/mTOR dual kinase inhibitor PF-04979064.

Design and synthesis of novel N-hydroxy-dihydronaphthyridinones as potent and orally bioavailable HIV-1 integrase inhibitors.

HIV-1 integrase (IN) is one of three enzymes encoded by the HIV genome and is essential for viral replication, and HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Recently, we reported the synthesis of orally bioavailable azaindole hydroxamic acids that were potent inhibitors of the HIV-1 IN enzyme. Here we disclose the design and synthesis of novel tricyclic N-hydroxy-dihydronaphthyridinones as potent, orally bioavailable HIV-1 integrase inhibitors displaying excellent ligand and lipophilic efficiencies.

Azaindole N-methyl hydroxamic acids as HIV-1 integrase inhibitors-II. The impact of physicochemical properties on ADME and PK.

HIV-1 integrase is one of three enzymes encoded by the HIV genome and is essential for viral replication, and HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Recently, we reported the discovery of azaindole hydroxamic acids that were potent inhibitors of the HIV-1 IN enzyme. N-Methyl hydroxamic acids were stable against oxidative metabolism, however were cleared rapidly through phase 2 glucuronidation pathways.

Azaindole hydroxamic acids are potent HIV-1 integrase inhibitors.

HIV-1 integrase (IN) is one of three enzymes encoded by the HIV genome and is essential for viral replication. Recently, HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Herein, we report the discovery of azaindole carboxylic acids and azaindole hydroxamic acids as potent inhibitors of the HIV-1 IN enzyme and their structure-activity relationships.