Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry.

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

Implementation of the CYP Index for the Design of Selective Tryptophan-2,3-dioxygenase Inhibitors.

A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation was demonstrated in vivo, which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the risk of drug-drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor containing a Lewis basic motif capable of binding heme.

Pharmacological Induction of RAS-GTP Confers RAF Inhibitor Sensitivity in KRAS Mutant Tumors.

Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism. Broad cell line profiling with RAF/MEK inhibitor combinations revealed synergistic efficacy in KRAS mutant and wild-type tumors, with KRASG13D mutants exhibiting greater synergy versus KRASG12 mutant tumors. Mechanistic studies demonstrate that MEK inhibition induced RAS-GTP levels, RAF dimerization and RAF kinase activity resulting in MEK phosphorylation in synergistic tumor lines regardless of KRAS status. Taken together, our studies uncover a strategy to rewire KRAS mutant tumors to confer sensitivity to RAF kinase inhibition.

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.

Chemically Diverse Group I p21-Activated Kinase (PAK) Inhibitors Impart Acute Cardiovascular Toxicity with a Narrow Therapeutic Window.

p21-activated kinase 1 (PAK1) has an important role in transducing signals in several oncogenic pathways. The concept of inhibiting this kinase has garnered significant interest over the past decade, particularly for targeting cancers associated with PAK1 amplification. Animal studies with the selective group I PAK (pan-PAK1, 2, 3) inhibitor G-5555 from the pyrido[2,3-d]pyrimidin-7-one class uncovered acute toxicity with a narrow therapeutic window. To attempt mitigating the toxicity, we introduced significant structural changes, culminating in the discovery of the potent pyridone side chain analogue G-9791. Mouse tolerability studies with this compound, other members of this series, and compounds from two structurally distinct classes revealed persistent toxicity and a correlation of minimum toxic concentrations and PAK1/2 mediated cellular potencies. Broad screening of selected PAK inhibitors revealed PAK1, 2, and 3 as the only overlapping targets. Our data suggest acute cardiovascular toxicity resulting from the inhibition of PAK2, which may be enhanced by PAK1 inhibition, and cautions against continued pursuit of pan-group I PAK inhibitors in drug discovery.

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.

Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pK a Polar Moiety.

Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.

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.

Structure-Guided Design of Group I Selective p21-Activated Kinase Inhibitors.

The p21-activated kinases (PAKs) play important roles in cytoskeletal organization, cellular morphogenesis, and survival and have generated significant attention as potential therapeutic targets for cancer. Following a high-throughput screen, we identified an aminopyrazole scaffold-based series that was optimized to yield group I selective PAK inhibitors. A structure-based design effort aimed at targeting the ribose pocket for both potency and selectivity led to much-improved group I vs II selectivity. Early lead compounds contained a basic primary amine, which was found to be a major metabolic soft spot with in vivo clearance proceeding predominantly via N-acetylation. We succeeded in identifying replacements with improved metabolic stability, leading to compounds with lower in vivo rodent clearance and excellent group I PAK selectivity.

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.

Learning and confirming with preclinical studies: modeling and simulation in the discovery of GDC-0917, an inhibitor of apoptosis proteins antagonist.

The application of modeling and simulation techniques is increasingly common in the preclinical stages of the drug development process. GDC-0917 [(S)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-N-(2-(oxazol-2-yl)-4-phenylthiazol-5-yl)pyrrolidine-2-carboxamide] is a potent second-generation antagonist of inhibitor of apoptosis (IAP) proteins that is being developed for the treatment of various cancers. GDC-0917 has low to moderate clearance in the mouse (12.0 ml/min/kg), rat (27.0 ml/min/kg), and dog (15.3 ml/min/kg), and high clearance in the monkey (67.6 ml/min/kg). Accordingly, oral bioavailability was lowest in monkeys compared with other species. Based on our experience with a prototype molecule with similar structure, in vitro-in vivo extrapolation was used to predict a moderate clearance (11.5 ml/min/kg) in humans. The predicted human volume of distribution was estimated using simple allometry at 6.69 l/kg. Translational pharmacokinetic-pharmacodynamic (PK-PD) analysis using results from MDA-MB-231-X1.1 breast cancer xenograft studies and predicted human pharmacokinetics suggests that ED50 and ED90 targets can be achieved in humans using acceptable doses (72 mg and 660 mg, respectively) and under an acceptable time frame. The relationship between GDC-0917 concentrations and pharmacodynamic response (cIAP1 degradation) was characterized using an in vitro peripheral blood mononuclear cell immunoassay. Simulations of human GDC-0917 plasma concentration-time profile and cIAP1 degradation at the 5-mg starting dose in the phase 1 clinical trial agreed well with observations. This work shows the importance of leveraging information from prototype molecules and illustrates how modeling and simulation can be used to add value to preclinical studies in the early stages of the drug development process.

SAHA enhances synaptic function and plasticity in vitro but has limited brain availability in vivo and does not impact cognition.

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer's disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology.

Noncovalent wild-type-sparing inhibitors of EGFR T790M.

UNLABELLED: Approximately half of EGFR-mutant non-small cell lung cancer (NSCLC) patients treated with small-molecule EGFR kinase inhibitors develop drug resistance associated with the EGF receptor (EGFR) T790M "gatekeeper" substitution, prompting efforts to develop covalent EGFR inhibitors, which can effectively suppress EGFR T790M in preclinical models. However, these inhibitors have yet to prove clinically efficacious, and their toxicity in skin, reflecting activity against wild-type EGFR, may limit dosing required to effectively suppress EGFR T790M in vivo. While profiling sensitivity to various kinase inhibitors across a large cancer cell line panel, we identified indolocarbazole compounds, including a clinically well-tolerated FLT3 inhibitor, as potent and reversible inhibitors of EGFR T790M that spare wild-type EGFR. These findings show the use of broad cancer cell profiling of kinase inhibitor efficacy to identify unanticipated novel applications, and they identify indolocarbazole compounds as potentially effective EGFR inhibitors in the context of T790M-mediated drug resistance in NSCLC. SIGNIFICANCE: EGFR-mutant lung cancer patients who respond to currently used EGFR kinase inhibitors invariably develop drug resistance, which is associated with the EGFR T790M resistance mutation in about half these cases. We unexpectedly identified a class of reversible potent inhibitors of EGFR T790M that do not inhibit wild-type EGFR, revealing a promising therapeutic strategy to overcome T790M-associated drug-resistant lung cancers.

Antitumor activity of targeted and cytotoxic agents in murine subcutaneous tumor models correlates with clinical response.

PURPOSE: Immunodeficient mice transplanted with subcutaneous tumors (xenograft or allograft) are widely used as a model of preclinical activity for the discovery and development of anticancer drug candidates. Despite their widespread use, there is a widely held view that these models provide minimal predictive value for discerning clinically active versus inactive agents. To improve the predictive nature of these models, we have carried out a retrospective population pharmacokinetic-pharmacodynamic (PK-PD) analysis of relevant xenograft/allograft efficacy data for eight agents (molecularly targeted and cytotoxic) with known clinical outcome. EXPERIMENTAL DESIGN: PK-PD modeling was carried out to first characterize the relationship between drug concentration and antitumor activity for each agent in dose-ranging xenograft or allograft experiments. Next, simulations of tumor growth inhibition (TGI) in xenografts/allografts at clinically relevant doses and schedules were carried out by replacing the murine pharmacokinetics, which were used to build the PK-PD model with human pharmacokinetics obtained from literature to account for species differences in pharmacokinetics. RESULTS: A significant correlation (r = 0.91, P = 0.0008) was observed between simulated xenograft/allograft TGI driven by human pharmacokinetics and clinical response but not when TGI observed at maximum tolerated doses in mice was correlated with clinical response (r = 0.36, P = 0.34). CONCLUSIONS: On the basis of these analyses, agents that led to greater than 60% TGI in preclinical models, at clinically relevant exposures, are more likely to lead to responses in the clinic. A proposed strategy for the use of murine subcutaneous models for compound selection in anticancer drug discovery is discussed.

Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152).

A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with K(i) values of 28, 14, 17, and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials, and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg, and the volume of distribution was 0.6 ± 0.2 L/kg.

Pharmacokinetic-pharmacodynamic analysis of vismodegib in preclinical models of mutational and ligand-dependent Hedgehog pathway activation.

PURPOSE: Vismodegib (GDC-0449) is a potent and selective inhibitor of the Hedgehog (Hh) pathway that shows antitumor activity in preclinical models driven by mutational or ligand-dependent activation of the Hh pathway. We wished to characterize the pharmacokinetic-pharmacodynamic (PK/PD) relationship of vismodegib in both model systems to guide optimal dose and schedule for vismodegib in the clinic. EXPERIMENTAL DESIGN: Preclinical efficacy and PK/PD studies were carried out with vismodegib in a Ptch(+/-) allograft model of medulloblastoma exhibiting mutational activation of the Hh pathway and patient-derived colorectal cancer (CRC) xenograft models exhibiting ligand-dependent pathway activation. Inhibition of the hedgehog pathway was related to vismodegib levels in plasma and to antitumor efficacy using an integrated population-based PK/PD model. RESULTS: Oral dosing of vismodegib caused tumor regressions in the Ptch(+/-) allograft model of medulloblastoma at doses ≥25 mg/kg and tumor growth inhibition at doses up to 92 mg/kg dosed twice daily in two ligand-dependent CRC models, D5123, and 1040830. Analysis of Hh pathway activity and PK/PD modeling reveals that vismodegib inhibits Gli1 with a similar IC(50) in both the medulloblastoma and D5123 models (0.165 µmol/L ±11.5% and 0.267 µmol/L ±4.83%, respectively). Pathway modulation was linked to efficacy using an integrated PK/PD model revealing a steep relationship where > 50% of the activity of vismodegib is associated with >80% repression of the Hh pathway. CONCLUSIONS: These results suggest that even small reductions in vismodegib exposure can lead to large changes in antitumor activity and will help guide proper dose selection for vismodegib in the clinic.

GDC-0449-a potent inhibitor of the hedgehog pathway.

SAR for a wide variety of heterocyclic replacements for a benzimidazole led to the discovery of functionalized 2-pyridyl amides as novel inhibitors of the hedgehog pathway. The 2-pyridyl amides were optimized for potency, PK, and drug-like properties by modifications to the amide portion of the molecule resulting in 31 (GDC-0449). Amide 31 produced complete tumor regression at doses as low as 12.5mg/kg BID in a medulloblastoma allograft mouse model that is wholly dependent on the Hh pathway for growth and is currently in human clinical trials, where it is initially being evaluated for the treatment of BCC.