High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRASG12C by Intact Protein MS and Targeted MRM.

With recent advances and success in several drugs designed to treat acute and chronic diseases, targeted covalent inhibitors show a resurgence in drug discovery. As covalent inhibition is time-dependent, the preferred quantitative potency metric of irreversible inhibitors is the second-order rate constant kinact/Ki, rather than IC50. Here, we present the development of a mass spectrometry-based platform for rapid kinetic analysis of irreversible covalent inhibitors. Using a simple liquid handling robot for automated sample preparation and a solid-phase extraction-based RapidFire-MS system for rapid MS analysis, kinetic characterization of covalent inhibitors was performed in high throughput both by intact protein analysis and targeted multiple reaction monitoring (MRM). In addition, a bimolecular reaction model was applied to extract kinact/Ki in data fitting, providing tremendous flexibility in the experimental design to characterize covalent inhibitors with various properties. Using KRASG12C inhibitors as a test case, the platform was demonstrated to be effective for studying covalent inhibitors with a wide range of kinact/Ki values from single digit to 3 × 105 M-1 s-1.

Development of a mass spectrometry-based tryptophan 2, 3-dioxygenase assay using liver cytosol from multiple species.

A novel and rapid method to determine the potency of inhibitors for tryptophan 2, 3-dioxygenase (TDO2) activities in human and preclinical species was successfully developed and validated utilizing LC-MS/MS. Previously reported TDO2 activity assays are resource intensive, requiring cloning and overexpression of TDO2. Here, we demonstrated that liver cytosol contained sufficient active TDO2 for evaluating the potency of TDO2 inhibitors across multiple species. TDO2 expression in human cytosol was estimated by LC-MS/MS to be 41 pmoL/mg cytosolic protein, with similar levels in dogs and monkeys, whereas mice and rats had 9.6 and 5.0-fold greater expression, respectively. Reaction conditions for TDO2-mediated conversion of l-tryptophan to kynurenine were optimized. Marked differences in kinetic parameters and inhibition potency were observed in TDO2 across species, with different Km values in dog (0.055 mM), monkey (0.070 mM), human (0.19 mM), mouse (0.32 mM) and rat (0.36 mM). Subsequently, IC50 values were determined for a series of TDO2 inhibitors in liver cytosol of five species, and good agreement with the literature values was observed for human enzyme. Taken together, these data indicate that TDO2 inhibition can be rapidly determined in readily available hepatic cytosol to assess potential species differences in potency.

An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells.

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

A mutation in APP protects against Alzheimer's disease and age-related cognitive decline.

The prevalence of dementia in the Western world in people over the age of 60 has been estimated to be greater than 5%, about two-thirds of which are due to Alzheimer's disease. The age-specific prevalence of Alzheimer's disease nearly doubles every 5 years after age 65, leading to a prevalence of greater than 25% in those over the age of 90 (ref. 3). Here, to search for low-frequency variants in the amyloid-ß precursor protein (APP) gene with a significant effect on the risk of Alzheimer's disease, we studied coding variants in APP in a set of whole-genome sequence data from 1,795 Icelanders. We found a coding mutation (A673T) in the APP gene that protects against Alzheimer's disease and cognitive decline in the elderly without Alzheimer's disease. This substitution is adjacent to the aspartyl protease ß-site in APP, and results in an approximately 40% reduction in the formation of amyloidogenic peptides in vitro. The strong protective effect of the A673T substitution against Alzheimer's disease provides proof of principle for the hypothesis that reducing the ß-cleavage of APP may protect against the disease. Furthermore, as the A673T allele also protects against cognitive decline in the elderly without Alzheimer's disease, the two may be mediated through the same or similar mechanisms.

From a novel HTS hit to potent, selective, and orally bioavailable KDM5 inhibitors.

A high-throughput screening (HTS) of the Genentech/Roche library identified a novel, uncharged scaffold as a KDM5A inhibitor. Lacking insight into the binding mode, initial attempts to improve inhibitor potency failed to improve potency, and synthesis of analogs was further hampered by the presence of a C-C bond between the pyrrolidine and pyridine. Replacing this with a C-N bond significantly simplified synthesis, yielding pyrazole analog 35, of which we obtained a co-crystal structure with KDM5A. Using structure-based design approach, we identified 50 with improved biochemical, cell potency and reduced MW and lower lipophilicity (LogD) compared with the original hit. Furthermore, 50 showed lower clearance than 9 in mice. In combination with its remarkably low plasma protein binding (PPB) in mice (40%), oral dosing of 50 at 5mg/kg resulted in unbound Cmax ∼2-fold of its cell potency (PC9 H3K4Me3 0.96µM), meeting our criteria for an in vivo tool compound from a new scaffold.

Lead optimization of a pyrazolo[1,5-a]pyrimidin-7(4H)-one scaffold to identify potent, selective and orally bioavailable KDM5 inhibitors suitable for in vivo biological studies.

Starting with a lead [1,5-a]pyrimidin-7(4H)-one-containing molecule (1), we generated potent, selective and orally bioavailable KDM5 inhibitors. Using structure- and property-based approaches, we designed 48 with improved cell potency (PC9 H3K4Me3 EC50=0.34µM). Furthermore, 48 maintained suitable physiochemical properties and displayed an excellent pharmacokinetic (PK) profile in mice. When dosed orally in mice at 50mg/kg twice a day (BID), 48 showed an unbound maximal plasma concentration (Cmax) >15-fold over its cell EC50, thereby providing a robust chemical probe for studying KDM5 biological functions in vivo.

Design and evaluation of 1,7-naphthyridones as novel KDM5 inhibitors.

Features from a high throughput screening (HTS) hit and a previously reported scaffold were combined to generate 1,7-naphthyridones as novel KDM5 enzyme inhibitors with nanomolar potencies. These molecules exhibited high selectivity over the related KDM4C and KDM2B isoforms. An X-ray co-crystal structure of a representative molecule bound to KDM5A showed that these inhibitors are competitive with the co-substrate (2-oxoglutarate or 2-OG).

Identification of potent, selective KDM5 inhibitors.

This communication describes the identification and optimization of a series of pan-KDM5 inhibitors derived from compound 1, a hit initially identified against KDM4C. Compound 1 was optimized to afford compound 20, a 10nM inhibitor of KDM5A. Compound 20 is highly selective for the KDM5 enzymes versus other histone lysine demethylases and demonstrates activity in a cellular assay measuring the increase in global histone 3 lysine 4 tri-methylation (H3K4me3). In addition compound 20 has good ADME properties, excellent mouse PK, and is a suitable starting point for further optimization.

Molecular mechanisms of Alzheimer disease protection by the A673T allele of amyloid precursor protein.

Pathogenic mutations in the amyloid precursor protein (APP) gene have been described as causing early onset familial Alzheimer disease (AD). We recently identified a rare APP variant encoding an alanine-to-threonine substitution at residue 673 (A673T) that confers protection against development of AD (Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) Nature 488, 96-99). The Ala-673 residue lies within the ß-secretase recognition sequence and is part of the amyloid-ß (Aß) peptide cleavage product (position 2 of Aß). We previously demonstrated that the A673T substitution makes APP a less favorable substrate for cleavage by BACE1. In follow-up studies, we confirm that A673T APP shows reduced cleavage by BACE1 in transfected mouse primary neurons and in isogenic human induced pluripotent stem cell-derived neurons. Using a biochemical approach, we show that the A673T substitution modulates the catalytic turnover rate (V(max)) of APP by the BACE1 enzyme, without affecting the affinity (K(m)) of the APP substrate for BACE1. We also show a reduced level of Aß(1-42) aggregation with A2T Aß peptides, an observation not conserved in Aß(1-40) peptides. When combined in a ratio of 1:9 Aß(1-42)/Aß(1-40) to mimic physiologically relevant mixtures, A2T retains a trend toward slowed aggregation kinetics. Microglial uptake of the mutant Aß(1-42) peptides correlated with their aggregation level. Cytotoxicity of the mutant Aß peptides was not dramatically altered. Taken together, our findings demonstrate that A673T, a protective allele of APP, reproducibly reduces amyloidogenic processing of APP and also mildly decreases Aß aggregation. These effects could together have an additive or even synergistic impact on the risk of developing AD.

Identification of orally-bioavailable antagonists of the TRPV4 ion-channel.

Antagonists of the TRPV4 receptor were identified using a focused screen, followed by a limited optimization program. The leading compounds obtained from this exercise, RN-1665 23 and RN-9893 26, showed moderate oral bioavailability when dosed to rats. The lead molecule, RN-9893 26, inhibited human, rat and murine variants of TRPV4, and showed excellent selectivity over related TRP receptors, such as TRPV1, TRPV3 and TRPM8. The overall profile for RN-9893 may permit its use as a proof-of-concept probe for in vivo applications.

Structure-Based Design and Evaluation of Reversible KRAS G13D Inhibitors.

Oncogenic KRAS mutations were identified decades ago, yet the selective inhibition of specific KRAS mutant proteins represents an ongoing challenge. Recent progress has been made in targeting certain P-loop mutant proteins, in particular KRAS G12C, for which the covalent inhibition of the GDP state via the Switch II pocket is now a clinically validated strategy. Inhibition of other KRAS mutant proteins such as KRAS G13D, on the other hand, still requires clinical validation. The remoteness of the D13 residue relative to the Switch II pocket in combination with the solvent exposure and conformational flexibility of the D13 side chain, as well as the difficulties of targeting carboxylate residues covalently, renders this specific protein particularly challenging to target selectively. In this report, we describe the design and evaluation of potent and KRAS G13D-selective reversible inhibitors. Subnanomolar binding to the GDP state Switch II pocket and biochemical selectivity over WT KRAS are achieved by leveraging a salt bridge with D13.

Evaluation of an Outpatient Pharmacist Consult Service at a Large Academic Medical Center.

OBJECTIVE: To evaluate a novel outpatient pharmacist consult service in a large academic medical center. SETTING: Four outpatient pharmacies that are part of a large academic medical center. METHODS: An outpatient pharmacist consult order was created and embedded in the electronic medical record (EMR). Medical center providers utilized this consult order when identifying patients in need of specific services provided by outpatient pharmacists. Descriptive data about each individual consult was collected including number completed, type of service, and duration. Rate of accepted pharmacy recommendations and patient cost savings were also evaluated. A survey was administered at the completion of the study period to assess provider and pharmacist satisfaction with the service. Patient demographic information was collected for those who had a documented completed consult. RESULTS: A total of 193 consults were completed: 137 immunizations, 37 care affordability, 15 education, 3 polypharmacy and 1 OTC recommendation. 89% of completed consults took pharmacists 20 minutes or less to complete. Of completed care affordability consults (n=31), 55% of patients saved between $100 - $500 per medication fill. Of providers who completed a survey and utilized the service (n=12), 83.3% were extremely satisfied and 16.7% were satisfied with it. The provider acceptance rate of pharmacist's recommendations was 74%. CONCLUSION: Implementation of an outpatient pharmacist consult service provided an alternative method for the utilization of pharmacist provided MTM services in outpatient pharmacies at a large academic medical center. The service was well received by both providers and pharmacists.

Oxime derivatives related to AP18: Agonists and antagonists of the TRPA1 receptor.

AP18 1 was recently disclosed as an antagonist of the TRPA1 receptor by the research group of Patapoutian. However, no detailed structure-activity relationships around 1 have been disclosed. Thus, a small number of oximes related to AP18 were examined in order to characterize the determinants of TRPA1 activity. Congeners of AP18 were found to possess both agonist and antagonist activity, suggesting that AP18 may behave as a covalent antagonist of the TRPA1 ion-channel.

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.

Identification and characterization of novel TRPV4 modulators.

TRPV4, a close relative of the vanilloid receptor TRPV1, is activated by diverse modalities such as endogenous lipid ligands, hypotonicity, protein kinases and, possibly, mechanical inputs. While its multiple roles in vivo are being explored with KO mice and selective agonists, there is a dearth of selective antagonists available to examine TRPV4 function. Herein we detail the use of a focused library of commercial compounds in order to identify RN-1747 and RN-1734, a pair of structurally related small molecules endowed with TRPV4 agonist and antagonist properties, respectively. Their activities against human, rat and mouse TRPV4 were characterized using electrophysiology and intracellular calcium influx. Significantly, antagonist RN-1734 was observed to completely inhibit both ligand- and hypotonicity-activated TRPV4. In addition, RN-1734 was found to be selective for TRPV4 in a TRP selectivity panel including TRPV1, TRPV3 and TRPM8, and could thus be a valuable pharmacological probe for TRPV4 studies.

Aminoisoxazoles as Potent Inhibitors of Tryptophan 2,3-Dioxygenase 2 (TDO2).

Tryptophan 2,3-dioxygenase 2 (TDO2) catalyzes the conversion of tryptophan to the immunosuppressive metabolite kynurenine. TDO2 overexpression has been observed in a number of cancers; therefore, TDO inhibition may be a useful therapeutic intervention for cancers. We identified an aminoisoxazole series as potent TDO2 inhibitors from a high-throughput screen (HTS). An extensive medicinal chemistry effort revealed that both the amino group and the isoxazole moiety are important for TDO2 inhibitory activity. Computational modeling yielded a binding hypothesis and provided insight into the observed structure-activity relationships. The optimized compound 21 is a potent TDO2 inhibitor with modest selectivity over indolamine 2,3-dioxygenase 1 (IDO1) and with improved human whole blood stability.

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.

A novel NMDA receptor positive allosteric modulator that acts via the transmembrane domain.

Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission and are key nervous system drug targets. While diverse pharmacological tools have yielded insight into iGluR extracellular domain function, less is known about molecular mechanisms underlying the ion conduction gating process within the transmembrane domain (TMD). We have discovered a novel NMDAR positive allosteric modulator (PAM), GNE-9278, with a unique binding site on the extracellular surface of the TMD. Mutation of a single residue near the Lurcher motif on GluN1 M3 can convert GNE-9278 modulation from positive to negative, and replacing three AMPAR pre-M1 residues with corresponding NMDAR residues can confer GNE-9278 sensitivity to AMPARs. Modulation by GNE-9278 is state-dependent and significantly alters extracellular domain pharmacology. The unique properties and structural determinants of GNE-9278 reveal new modulatory potential of the iGluR TMD.

Selective Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12) with Activity in a Model of Alzheimer's Disease.

Significant data exists to suggest that dual leucine zipper kinase (DLK, MAP3K12) is a conserved regulator of neuronal degeneration following neuronal injury and in chronic neurodegenerative disease. Consequently, there is considerable interest in the identification of DLK inhibitors with a profile compatible with development for these indications. Herein, we use structure-based drug design combined with a focus on CNS drug-like properties to generate compounds with superior kinase selectivity and metabolic stability as compared to previously disclosed DLK inhibitors. These compounds, exemplified by inhibitor 14, retain excellent CNS penetration and are well tolerated following multiple days of dosing at concentrations that exceed those required for DLK inhibition in the brain.

Loss of dual leucine zipper kinase signaling is protective in animal models of neurodegenerative disease.

Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer's disease. We demonstrate that DLK/c-Jun N-terminal kinase signaling was increased in mouse models and human patients with these disorders and that genetic deletion of DLK protected against axon degeneration, neuronal loss, and functional decline in vivo. Furthermore, pharmacological inhibition of DLK activity was sufficient to attenuate the neuronal stress response and to provide functional benefit even in the presence of ongoing disease. These findings demonstrate that pathological activation of DLK is a conserved mechanism that regulates neurodegeneration and suggest that DLK inhibition may be a potential approach to treat multiple neurodegenerative diseases.