Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach.

KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C-positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/µg of total protein has been achieved for both free and GDC-6036-bound KRAS G12C with as little as 4 µg of total protein extracted from human tumor samples. This sub-fmol/µg level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.

Discovery of Spiro-azaindoline Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1).

Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1. Using a structure-based drug design approach, the kinase selectivity of the molecules was significantly improved by inducing and stabilizing an unusual P-loop folded binding mode. The metabolic liabilities of the initial 7-azaindole high-throughput screening hit were mitigated by addressing a key metabolic soft spot along with physicochemical property-based optimization. The resulting spiro-azaindoline HPK1 inhibitors demonstrated improved in vitro ADME properties and the ability to induce cytokine production in primary human T-cells.

RTK-Dependent Inducible Degradation of Mutant PI3Kα Drives GDC-0077 (Inavolisib) Efficacy.

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. SIGNIFICANCE: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors.See related commentary by Vanhaesebroeck et al., p. 20.This article is highlighted in the In This Issue feature, p. 1.

Characterization of Antineovascularization Activity and Ocular Pharmacokinetics of Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor GNE-947.

The objectives of the present study were to characterize GNE-947 for its phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitory activities, in vitro anti-cell migration activity in human umbilical vein endothelial cells (HUVECs), in vivo antineovascularization activity in laser-induced rat choroidal neovascular (CNV) eyes, pharmacokinetics in rabbit plasma and eyes, and ocular distribution using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) and autoradioluminography. Its PI3K and mTOR K i were 0.0005 and 0.045 µM, respectively, and its HUVEC IC50 was 0.093 µM. GNE-947 prevented neovascularization in the rat CNV model at 50 or 100 µg per eye with repeat dosing. After a single intravenous injection at 2.5 and 500 µg/kg in rabbits, its plasma terminal half-lives (t 1/2) were 9.11 and 9.59 hours, respectively. After a single intravitreal injection of a solution at 2.5 µg per eye in rabbits, its apparent t 1/2 values were 14.4, 16.3, and 23.2 hours in the plasma, vitreous humor, and aqueous humor, respectively. After a single intravitreal injection of a suspension at 33.5, 100, 200 µg per eye in rabbits, the t 1/2 were 29, 74, and 219 days in the plasma and 46, 143, and 191 days in the eyes, respectively. MALDI-IMS and autoradioluminography images show that GNE-947 did not homogenously distribute in the vitreous humor and aggregated at the injection sites after injection of the suspension, which was responsible for the long t 1/2 of the suspension because of the slow dissolution process. This hypothesis was supported by pharmacokinetic modeling analyses. In conclusion, the PI3K/mTOR inhibitor GNE-947 prevented neovascularization in a rat CNV model, with t 1/2 up to approximately 6 months after a single intravitreal injection of the suspension in rabbit eyes. SIGNIFICANCE STATEMENT: GNE-947 is a potent phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor and exhibits anti-choroidal neovascular activity in rat eyes. The duration of GNE-947 in the rabbit eyes after intravitreal injection in a solution is short, with a half-life (t 1/2) of less than a day. However, the duration after intravitreal dose of a suspension is long, with t 1/2 up to 6 months due to low solubility and slow dissolution. These results indicate that intravitreal injection of a suspension for low-solubility drugs can be used to achieve long-term drug exposure.

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma.

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKa = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uu = 0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.

Hematopoietic Progenitor Kinase-1 Structure in a Domain-Swapped Dimer.

Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.

Challenges of developing small-molecule kinase inhibitors for brain tumors and the need for emphasis on free drug levels.

Despite biological rationale and significant clinical study, the pursuit of small-molecule kinase inhibitors for the treatment of brain cancers has had very limited success. This Advance-in-Brief discusses the need for drugs to achieve free brain penetration to engage their targets where CNS tumors reside. This need to achieve free, as opposed to total, drug concentrations in the brain may be a contributing factor to why so many small-molecule kinase inhibitors have not realized success in the neuro-oncology setting. For kinase targets of interest for brain cancer, either the vast majority of small-molecule inhibitors have data suggesting that free brain penetration would be limited or there are inadequate data to suggest that free brain penetration could be expected. Therefore, kinase targets of interest in the treatment of brain cancers may be inadequately assessed due to a lack of freely brain-penetrant inhibitors available for clinical study. Encouraging recent drug discovery efforts that focused on achieving free brain penetration for cancers in the CNS are highlighted. Still, further efforts are needed to enable thorough clinical evaluation of biological hypotheses.

CNS Anticancer Drug Discovery and Development: 2016 conference insights.

CNS Anticancer Drug Discovery and Development, 16-17 November 2016, Scottsdale, AZ, USA The 2016 second CNS Anticancer Drug Discovery and Development Conference addressed diverse viewpoints about why new drug discovery/development focused on CNS cancers has been sorely lacking. Despite more than 70,000 individuals in the USA being diagnosed with a primary brain malignancy and 151,669-286,486 suffering from metastatic CNS cancer, in 1999, temozolomide was the last drug approved by the US FDA as an anticancer agent for high-grade gliomas. Among the topics discussed were economic factors and pharmaceutical risk assessments, regulatory constraints and perceptions and the need for improved imaging surrogates of drug activity. Included were modeling tumor growth and drug effects in a medical environment in which direct tumor sampling for biological effects can be problematic, potential new drugs under investigation and targets for drug discovery and development. The long trajectory and diverse impediments to novel drug discovery, and expectation that more than one drug will be needed to adequately inhibit critical intracellular tumor pathways were viewed as major disincentives for most pharmaceutical/biotechnology companies. While there were a few unanimities, one consensus is the need for continued and focused discussion among academic and industry scientists and clinicians to address tumor targets, new drug chemistry, and more time- and cost-efficient clinical trials based on surrogate end points.

Brain Distribution and Efficacy of the Brain Penetrant PI3K Inhibitor GDC-0084 in Orthotopic Mouse Models of Human Glioblastoma.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Limited treatment options have only marginally impacted patient survival over the past decades. The phophatidylinositol 3-kinase (PI3K) pathway, frequently altered in GBM, represents a potential target for the treatment of this glioma. 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) is a PI3K inhibitor that was specifically optimized to cross the blood-brain barrier. The goals of our studies were to characterize the brain distribution, pharmacodynamic (PD) effect, and efficacy of GDC-0084 in orthotopic xenograft models of GBM. GDC-0084 was tested in vitro to assess its sensitivity to the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and in vivo in mice to evaluate its effects on the PI3K pathway in intact brain. Mice bearing U87 or GS2 intracranial tumors were treated with GDC-0084 to assess its brain distribution by matrix-assisted laser desorption ionization (MALDI) imaging and measure its PD effects and efficacy in GBM orthotopic models. Studies in transfected cells indicated that GDC-0084 was not a substrate of P-gp or BCRP. GDC-0084 markedly inhibited the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. MALDI imaging showed GDC-0084 distributed evenly in brain and intracranial U87 and GS2 tumors. GDC-0084 achieved significant tumor growth inhibition of 70% and 40% against the U87 and GS2 orthotopic models, respectively. GDC-0084 distribution throughout the brain and intracranial tumors led to potent inhibition of the PI3K pathway. Its efficacy in orthotopic models of GBM suggests that it could be effective in the treatment of GBM. GDC-0084 is currently in phase I clinical trials.

Discovery of a Noncovalent, Mutant-Selective Epidermal Growth Factor Receptor Inhibitor.

Inhibitors targeting the activating mutants of the epidermal growth factor receptor (EGFR) have found success in the treatment of EGFR mutant positive non-small-cell lung cancer. A secondary point mutation (T790M) in the inhibitor binding site has been linked to the acquired resistance against those first generation therapeutics. Herein, we describe the lead optimization of a series of reversible, pan-mutant (L858R, del746-750, T790M/L858R, and T790M/del746-750) EGFR inhibitors. By use of a noncovalent double mutant (T790M/L858R and T790M/del746-750) selective EGFR inhibitor (2) as a starting point, activities against the single mutants (L858R and del746-750) were introduced through a series of structure-guided modifications. The in vitro ADME-PK properties of the lead molecules were further optimized through a number of rational structural changes. The resulting inhibitor (21) exhibited excellent cellular activity against both the single and double mutants of EGFR, demonstrating target engagement in vivo and ADME-PK properties that are suitable for further evaluation. The reversible, noncovalent inhibitors described complement the covalent pan-mutant EGFR inhibitors that have shown encouraging results in recent clinical trials.

Small Molecule Kinase Inhibitors for the Treatment of Brain Cancer.

In addition to each of the factors that govern the identification of a successful oncology drug candidate, drug discovery aimed at treating neurological cancer must also consider the presence of the blood-brain barrier (BBB). The high level of expression of efflux transporters (e.g., P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp)) at the BBB limits many small molecules from freely reaching the brain, where neurooncologic malignancies reside. Furthermore, many of the targets identified for the potential treatment of central nervous system (CNS) malignancies suggest that kinase inhibitors, capable of penetrating the BBB to reach their target, would be desirable. This Perspective discusses the unmet need for neurooncology treatments, the appeal of kinase targets in this space, and a summary of what is known about free brain penetration of clinical inhibitors of kinases that are of interest for the treatment of brain cancer.

Discovery of Clinical Development Candidate GDC-0084, a Brain Penetrant Inhibitor of PI3K and mTOR.

Inhibition of phosphoinositide 3-kinase (PI3K) signaling is an appealing approach to treat brain tumors, especially glioblastoma multiforme (GBM). We previously disclosed our successful approach to prospectively design potent and blood-brain barrier (BBB) penetrating PI3K inhibitors. The previously disclosed molecules were ultimately deemed not suitable for clinical development due to projected poor metabolic stability in humans. We, therefore, extended our studies to identify a BBB penetrating inhibitor of PI3K that was also projected to be metabolically stable in human. These efforts required identification of a distinct scaffold for PI3K inhibitors relative to our previous efforts and ultimately resulted in the identification of GDC-0084 (16). The discovery and preclinical characterization of this molecule are described within.

Pyridones as Highly Selective, Noncovalent Inhibitors of T790M Double Mutants of EGFR.

The rapid advancement of a series of noncovalent inhibitors of T790M mutants of EGFR is discussed. The optimization of pyridone 1, a nonselective high-throughput screening hit, to potent molecules with high levels of selectivity over wtEGFR and the broader kinome is described herein.

The Rational Design of Selective Benzoxazepin Inhibitors of the α-Isoform of Phosphoinositide 3-Kinase Culminating in the Identification of (S)-2-((2-(1-Isopropyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)oxy)propanamide (GDC-0326).

Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy. Despite the particular attraction of targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other class I isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously minimized inhibition of PI3Kß relative to the other class I insoforms. Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a nonconserved residue. Several molecules selective for PI3Kα relative to the other class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4).

4-Aminoindazolyl-dihydrofuro[3,4-d]pyrimidines as non-covalent inhibitors of mutant epidermal growth factor receptor tyrosine kinase.

The treatment of epidermal growth factor receptor (EGFR)-driven non-small cell lung cancers with the T790M resistance mutation remains a significant unmet medical need. We report the identification of 4-aminoindazolyl-dihydrofuro[3,4-d]pyrimidines as non-covalent inhibitors of EGFR, with excellent activity against the T790M resistance double mutants and initial single activating mutants. Using an optimization strategy focused on structure-based design and improving PK properties through metabolite identification, we obtained advanced leads with high oral exposure.

Noncovalent Mutant Selective Epidermal Growth Factor Receptor Inhibitors: A Lead Optimization Case Study.

Because of their increased activity against activating mutants, first-generation epidermal growth factor receptor (EGFR) kinase inhibitors have had remarkable success in treating non-small-cell lung cancer (NSCLC) patients, but acquired resistance, through a secondary mutation of the gatekeeper residue, means that clinical responses only last for 8-14 months. Addressing this unmet medical need requires agents that can target both of the most common double mutants: T790M/L858R (TMLR) and T790M/del(746-750) (TMdel). Herein we describe how a noncovalent double mutant selective lead compound was optimized using a strategy focused on the structure-guided increase in potency without added lipophilicity or reduction of three-dimensional character. Following successive rounds of design and synthesis it was discovered that cis-fluoro substitution on 4-hydroxy- and 4-methoxypiperidinyl groups provided synergistic, substantial, and specific potency gain through direct interaction with the enzyme and/or effects on the proximal ligand oxygen atom. Further development of the fluorohydroxypiperidine series resulted in the identification of a pair of diastereomers that showed 50-fold enzyme and cell based selectivity for T790M mutants over wild-type EGFR (wtEGFR) in vitro and pathway knock-down in an in vivo xenograft model.

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. While this White Paper is not a formal roadmap to that end, it should be an educational guide to clinicians and scientists to help move a stagnant field forward.

A probabilistic method to report predictions from a human liver microsomes stability QSAR model: a practical tool for drug discovery.

Using data from the in vitro liver microsomes metabolic stability assay, we have developed QSAR models to predict in vitro human clearance. Models were trained using in house high-throughput assay data reported as the predicted human hepatic clearance by liver microsomes or pCLh. Machine learning regression methods were used to generate the models. Model output for a given molecule was reported as its probability of being metabolically stable, thus allowing for synthesis prioritization based on this prediction. Use of probability, instead of the regression value or categories, has been found to be an efficient way for both reporting and assessing predictions. Model performance is evaluated using prospective validation. These models have been integrated into a number of desktop tools, and the models are routinely used to prioritize the synthesis of compounds. We discuss two therapeutic projects at Genentech that exemplify the benefits of a probabilistic approach in applying the models. A three-year retrospective analysis of measured liver microsomes stability data on all registered compounds at Genentech reveals that the use of these models has resulted in an improved metabolic stability profile of synthesized compounds.

Discovery of selective and noncovalent diaminopyrimidine-based inhibitors of epidermal growth factor receptor containing the T790M resistance mutation.

Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties.