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Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target’s resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRASG12Cthat bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849as a potent, selective covalent inhibitor of KRASG12Cis described.

Mesenchymal-epithelial transition factor (MET) is a receptor tyrosine kinase that serves a critical function in numerous developmental, morphogenic, and proliferative signaling pathways. If dysregulated, MET has been shown to be involved in the development and survival of several cancers, including non-small cell lung cancer (NSCLC), renal cancer, and other epithelial tumors. Currently, the clinical efficacy of FDA approved MET inhibitors is limited by on-target acquired resistance, dose-limiting toxicities, and less than optimal efficacy against brain metastasis. Therefore, there is still an unmet medical need for the development of MET inhibitors to address these issues. Herein we report the application of structure-based design for the discovery and development of a novel class of brain-penetrant MET inhibitors with enhanced activity against clinically relevant mutations and improved selectivity. Compound 13with a MET D1228N cell line IC50value of 23 nM showed good efficacy in an intracranial tumor model and increased the median overall survival of the animals to 100% when dosed orally at 100 mg/kg daily for 21 days.

GABAAreceptors (GABAARs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([3H]azietomidate) and mephobarbital [[3H]1-methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid ([3H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the α1β3γ2 GABAAR transmembrane domain at β+–α−(β+sites) and α+–β−/γ+–β−(β−sites) subunit interfaces. We now use competition photolabeling with [3H]azietomidate and [3H]R-mTFD-MPAB to identify para-substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to β+, while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to β−sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the α+–β−and γ+–β−sites. However, we discovered four compounds that bind with different affinities to the two β−interface sites. Two of these bind with higher affinity to one of the β−sites than to the β+sites. We deduce that 4-benzoyl-propofol binds with >100-fold higher affinity to the γ+–β−site than to the α+–β−or β+–α−sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the α+–β−site than to the β+and γ+–β−sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABAAR transmembrane domain, a property that may facilitate the development of subtype selective GABAAR PAMs.

At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAAreceptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [3H]R-mTFD-MPAB ([3H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo(muscle-type) nAChR. For nAChRs expressed in Xenopusoocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [3H]ACh to nAChR-rich membranes (EC50= 9 µM) and inhibited binding of the ion channel blocker [3H]tenocyclidine in the nAChR desensitized and resting states with IC50values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [3H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ–αsubunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ–αsubunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.