Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2.

Conformational selection by small molecules expands inhibitory possibilities for protein kinases. Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation induces global motions involving exchange between two states, L and R. We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analog. Thus, the L→R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors.

RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.

Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.

Highly potent and selective 3-N-methylquinazoline-4(3H)-one based inhibitors of B-Raf(V600E) kinase.

Herein we describe the design of a novel series of ATP competitive B-Raf inhibitors via structure-based methods. These 3-N-methylquinazoline-4(3H)-one based inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 16, a potent, selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth inhibition in xenograft studies. Our work also demonstrates that by replacing an aryl amide with an aryl sulfonamide, a multikinase inhibitor such as AZ-628, can be converted to a selective B-Raf inhibitor, a finding that should have broad application in kinase drug discovery.

Synthesis, characterization, and PK/PD studies of a series of spirocyclic pyranochromene BACE1 inhibitors.

The development of 1,3,4,4a,5,10a-hexahydropyrano[3,4-b]chromene analogs as BACE1 inhibitors is described. Introduction of the spirocyclic pyranochromene scaffold yielded several advantages over previous generation cores, including increased potency, reduced efflux, and reduced CYP2D6 inhibition. Compound 13 (BACE1 IC50=110 nM) demonstrated a reduction in CSF Aß in wild type rats after a single dose.

Conformation selection by ATP-competitive inhibitors and allosteric communication in ERK2.

Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named 'L' and 'R,' where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

Conformation Selection by ATP-competitive Inhibitors and Allosteric Communication in ERK2.

Activation of the extracellular signal regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named "L" and "R", where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12D Inhibitor.

KRASG12D, the most common oncogenic KRAS mutation, is a promising target for the treatment of solid tumors. However, when compared to KRASG12C, selective inhibition of KRASG12D presents a significant challenge due to the requirement of inhibitors to bind KRASG12D with high enough affinity to obviate the need for covalent interactions with the mutant KRAS protein. Here, we report the discovery and characterization of the first noncovalent, potent, and selective KRASG12D inhibitor, MRTX1133, which was discovered through an extensive structure-based activity improvement and shown to be efficacious in a KRASG12D mutant xenograft mouse tumor model.

Non-oxime inhibitors of B-Raf(V600E) kinase.

The development of inhibitors of B-Raf(V600E) serine-threonine kinase is described. Various head-groups were examined to optimize inhibitor activity and ADME properties. Several of the head-groups explored, including naphthol, phenol and hydroxyamidine, possessed good activity but had poor pharmacokinetic exposure in mice. Exposure was improved by incorporating more metabolically stable groups such as indazole and tricyclic pyrazole, while indazole could also be optimized for good cellular activity.

Discovery and SAR of spirochromane Akt inhibitors.

A novel series of spirochromane pan-Akt inhibitors is reported. SAR optimization furnished compounds with improved enzyme potencies and excellent selectivity over the related AGC kinase PKA. Attempted replacement of the phenol hinge binder provided compounds with excellent Akt enzyme and cell activities but greatly diminished selectivity over PKA.

The discovery of furo[2,3-c]pyridine-based indanone oximes as potent and selective B-Raf inhibitors.

Virtual and high-throughput screening identified imidazo[1,2-a]pyrazines as inhibitors of B-Raf. We describe the rationale, SAR, and evolution of the initial hits to a series of furo[2,3-c]pyridine indanone oximes as highly potent and selective inhibitors of B-Raf.

Non-oxime pyrazole based inhibitors of B-Raf kinase.

The synthesis and biological evaluation of non-oxime pyrazole based B-Raf inhibitors is reported. Several oxime replacements have been prepared and have shown excellent enzyme activity. Further optimization of fused pyrazole 2a led to compound 38, a selective and potent B-Raf inhibitor.

Discovery of spirocyclic sulfonamides as potent Akt inhibitors with exquisite selectivity against PKA.

We describe the design and synthesis of novel bicyclic spiro sulfonamides as potent Akt inhibitors. Through structure-based rational design, we have successfully improved PKA selectivity of previously disclosed spirochromanes. Representative compounds showed favorable Akt potency while exhibiting up to 1000-fold selectivity against PKA.

Discovery of pyrrolopyrimidine inhibitors of Akt.

The discovery and optimization of a series of pyrrolopyrimidine based protein kinase B (Pkb/Akt) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent inhibition of all three Akt isoforms and knockdown of phospho-PRAS40 levels in LNCaP cells and tumor xenografts.

Discovery of dihydrothieno- and dihydrofuropyrimidines as potent pan Akt inhibitors.

Herein we report the discovery and synthesis of a novel series of dihydrothieno- and dihydrofuropyrimidines (2 and 3) as potent pan Akt inhibitors. Utilizing previous SAR and analysis of the amino acid sequences in the binding site we have designed inhibitors displaying increased PKA and general kinase selectivity with improved tolerability compared to the progenitor pyrrolopyrimidine (1). A representative dihydrothieno compound (34) was advanced into a PC3-NCI prostate mouse tumor model in which it demonstrated a dose-dependent reduction in tumor growth and stasis when dosed orally daily at 200 mg/kg.

Identification of the Clinical Development Candidate MRTX849, a Covalent KRASG12C Inhibitor for the Treatment of Cancer.

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 KRASG12C that 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 MRTX849 as a potent, selective covalent inhibitor of KRASG12C is described.

The KRASG12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients.

Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties, selectively modifies mutant cysteine 12 in GDP-bound KRASG12C, and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) KRASG12C-positive cell line- and patient-derived xenograft models from multiple tumor types, and objective responses have been observed in patients with KRASG12C-positive lung and colon adenocarcinomas. Comprehensive pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant nonclinical models identified mechanisms implicated in limiting antitumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of receptor tyrosine kinases (RTK), bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models. SIGNIFICANCE: The discovery of MRTX849 provides a long-awaited opportunity to selectively target KRASG12C in patients. The in-depth characterization of MRTX849 activity, elucidation of response and resistance mechanisms, and identification of effective combinations provide new insight toward KRAS dependence and the rational development of this class of agents.See related commentary by Klempner and Hata, p. 20.This article is highlighted in the In This Issue feature, p. 1.

Potent and selective pyrazole-based inhibitors of B-Raf kinase.

Herein we describe a novel pyrazole-based class of ATP competitive B-Raf inhibitors. These inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. A subset of these inhibitors has demonstrated the ability to inhibit downstream ERK phosphorylation in LOX tumors from mouse xenograft studies.

Non-charged thiamine analogs as inhibitors of enzyme transketolase.

Inhibition of the thiamine-utilizing enzyme transketolase (TK) has been linked with diminished tumor cell proliferation. Most thiamine antagonists have a permanent positive charge on the B-ring, and it has been suggested that this charge is required for diphosphorylation by thiamine pyrophosphokinase (TPPK) and binding to TK. We sought to make neutral thiazolium replacements that would be substrates for TPPK, while not necessarily needing thiamine transporters (ThTr1 and ThTr2) for cell penetration. The synthesis, SAR, and structure-based rationale for highly potent non-thiazolium TK antagonists are presented.

Synthesis, in vitro and in vivo activity of thiamine antagonist transketolase inhibitors.

Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.

Discovery of Tetrahydropyridopyrimidines as Irreversible Covalent Inhibitors of KRAS-G12C with In Vivo Activity.

KRAS is the most frequently mutated driver oncogene in human cancer, and KRAS mutations are commonly associated with poor prognosis and resistance to standard treatment. The ability to effectively target and block the function of mutated KRAS has remained elusive despite decades of research. Recent findings have demonstrated that directly targeting KRAS-G12C with electrophilic small molecules that covalently modify the mutated codon 12 cysteine is feasible. We have discovered a series of tetrahydropyridopyrimidines as irreversible covalent inhibitors of KRAS-G12C with in vivo activity. The PK/PD and efficacy of compound 13 will be highlighted.