The reactivity-driven biochemical mechanism of covalent KRASG12C inhibitors.

Activating mutations in KRAS are among the most common tumor driver mutations. Until recently, KRAS had been considered undruggable with small molecules; the discovery of the covalent KRASG12C inhibitors ARS-853 and ARS-1620 has demonstrated that it is feasible to inhibit KRAS with high potency in cells and animals. Although the biological activity of these inhibitors has been described, the biochemical mechanism of how the compounds achieve potent inhibition remained incompletely understood. We now show that the activity of ARS-853 and ARS-1620 is primarily driven by KRAS-mediated catalysis of the chemical reaction with Cys12 in human KRASG12C, while the reversible binding affinity is weak, in the hundreds of micromolar or higher range. The mechanism resolves how an induced, shallow and dynamic pocket not expected to support high-affinity binding of small molecules can nevertheless be targeted with potent inhibitors and may be applicable to other targets conventionally considered undruggable.

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State.

UNLABELLED: KRAS gain-of-function mutations occur in approximately 30% of all human cancers. Despite more than 30 years of KRAS-focused research and development efforts, no targeted therapy has been discovered for cancers with KRAS mutations. Here, we describe ARS-853, a selective, covalent inhibitor of KRAS(G12C) that inhibits mutant KRAS-driven signaling by binding to the GDP-bound oncoprotein and preventing activation. Based on the rates of engagement and inhibition observed for ARS-853, along with a mutant-specific mass spectrometry-based assay for assessing KRAS activation status, we show that the nucleotide state of KRAS(G12C) is in a state of dynamic flux that can be modulated by upstream signaling factors. These studies provide convincing evidence that the KRAS(G12C) mutation generates a "hyperexcitable" rather than a "statically active" state and that targeting the inactive, GDP-bound form is a promising approach for generating novel anti-RAS therapeutics. SIGNIFICANCE: A cell-active, mutant-specific, covalent inhibitor of KRAS(G12C) is described that targets the GDP-bound, inactive state and prevents subsequent activation. Using this novel compound, we demonstrate that KRAS(G12C) oncoprotein rapidly cycles bound nucleotide and responds to upstream signaling inputs to maintain a highly active state.

Using 'biased-privileged' scaffolds to identify lysine methyltransferase inhibitors.

Methylation of histones by lysine methyltransferases (KMTases) plays important roles in regulating chromatin function. It is also now clear that improper KMTases activity is linked to human diseases, such as cancer. We report an approach that employs drug-like 'privileged' scaffolds biased with motifs present in S-adenosyl methionine, the cofactor used by KMTases, to efficiently generate inhibitors for Set7, a biochemically well-characterized KMTase. Setin-1, the most potent inhibitor of Set7 we have developed also inhibits the KMTase G9a. Together these data suggest that these inhibitors should provide good starting points to generate useful probes for KMTase biology and guide the design of KMTase inhibitors with drug-like properties.

K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions.

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.

Staurosporine-derived inhibitors broaden the scope of analog-sensitive kinase technology.

Analog-sensitive (AS) kinase technology is a powerful approach for studying phospho-signaling pathways in diverse organisms and physiological processes. The key feature of this technique is that a kinase-of-interest can be mutated to sensitize it to inhibitor analogs that do not target wild-type (WT) kinases. In theory, this enables specific inhibition of any kinase in cells and in mouse models of human disease. Typically, these inhibitors are identified from a small library of molecules based on the pyrazolopyrimidine (PP) scaffold. However, we recently identified a subset of native human kinases, including the Ephrin A kinase family, that are sensitive to commonly used PP inhibitors. In an effort to develop a bioorthogonal AS-kinase inhibitor and to extend this technique to PP-sensitive kinases, we sought an alternative inhibitor scaffold. Here we report the structure-based design of synthetically tractable, potent, and extremely selective AS-kinase inhibitors based on the natural product staurosporine. We demonstrate that these molecules, termed staralogs, potently target AS kinases in cells, and we employ X-ray crystallography to elucidate their mechanism of efficacy. Finally, we demonstrate that staralogs target AS mutants of PP-sensitive kinases at concentrations where there is little to no inhibition of native human kinases. Thus, staralogs represent a new class of AS-kinase inhibitors and a core component of the chemical genetic tool kit for probing kinase-signaling pathways.

Chemical genetic strategy for targeting protein kinases based on covalent complementarity.

The conserved nature of the ATP-binding site of the > 500 human kinases renders the development of specific inhibitors a challenging task. A widely used chemical genetic strategy to overcome the specificity challenge exploits a large-to-small mutation of the gatekeeper residue (a conserved hydrophobic amino acid) and the use of a bulky inhibitor to achieve specificity via shape complementarity. However, in a number of cases, introduction of a glycine or alanine gatekeeper results in diminished kinase activity and ATP affinity. A new chemical genetic approach based on covalent complementarity between an engineered gatekeeper cysteine and an electrophilic inhibitor was developed to address these challenges. This strategy was evaluated with Src, a proto-oncogenic tyrosine kinase known to lose some enzymatic activity using the shape complementarity chemical genetic strategy. We found that Src with a cysteine gatekeeper recapitulates wild type activity and can be irreversibly inhibited both in vitro and in cells. A cocrystal structure of T338C c-Src with a vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor was solved to elucidate the inhibitor binding mode. A panel of electrophilic inhibitors was analyzed against 307 kinases and MOK (MAPK/MAK/MRK overlapping kinase), one of only two human kinases known to have an endogenous cysteine gatekeeper. This analysis revealed remarkably few off-targets, making these compounds the most selective chemical genetic inhibitors reported to date. Protein engineering studies demonstrated that it is possible to increase inhibitor potency through secondary-site mutations. These results suggest that chemical genetic strategies based on covalent complementarity should be widely applicable to the study of protein kinases.

Polo-like kinase controls vertebrate spindle elongation and cytokinesis.

During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.

Probing cell-division phenotype space and Polo-like kinase function using small molecules.

Cell-permeable small molecules that inhibit their targets on fast timescales are powerful probes of cell-division mechanisms. Such inhibitors have been identified using phenotype-based screens with chemical libraries. However, the characteristics of compound libraries needed to effectively span cell-division phenotype space, to find probes that target different mechanisms, are not known. Here we show that a small collection of 100 diaminopyrimidines (DAPs) yields a range of cell-division phenotypes, including changes in spindle geometry, chromosome positioning and mitotic index. Monopolar mitotic spindles are induced by four inhibitors, including one that targets Polo-like kinases (Plks), evolutionarily conserved serine/threonine kinases. Using chemical inhibitors and high-resolution live-cell microscopy, we found that Plk activity is needed for the assembly and maintenance of bipolar mitotic spindles. Plk inhibition destabilizes kinetochore microtubules while stabilizing other spindle microtubules, leading to monopolar spindles. Further testing of compounds based on 'privileged scaffolds', such as the DAP scaffold, could lead to new cell-division probes and antimitotic agents.

Evaluation of 8-arylsulfanyl, 8-arylsulfoxyl, and 8-arylsulfonyl adenine derivatives as inhibitors of the heat shock protein 90.

Hsp90 is a chaperone protein with important roles in maintaining transformation and in elevating the survival and growth potential of cancer cells. Currently there is an increasing interest in developing inhibitors of this protein as anticancer therapeutics. One of such inhibitors, the purine-scaffold class, has been reported to be potent and selective against Hsp90 both in vitro and in vivo models of cancer. Here, a series of 8-arylsulfanyl, -sulfoxyl, and -sulfonyl adenine members of the purine class was synthesized and evaluated as inhibitors of the chaperone. The structure-activity relationship and selectivity for tumor Hsp90 of compounds within the series is presented. Our results suggest that 8-arylsulfanyl adenine derivatives are good inhibitors of chaperone activity, whereas oxidation of the sulfides to sulfoxides or sulfones leads to compounds of decreased activity. The study identifies derivative 11v as the most potent Hsp90 inhibitor of the purine-scaffold series published to date (EC(50) = 30 nM), and also as the compound of this class with highest selectivity for tumor vs normal cell Hsp90 (700 to 3000-fold). Most rewardingly, this work has allowed for the identification of Hsp90 inhibitors with selective affinities for Hsp90-client protein complexes, derivatives that may represent useful pharmacological tools in dissecting Hsp90-regulated processes.

Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and -sensitive cancer cells.

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.

Three-component condensation leading to beta-amino acid diamides: convergent assembly of beta-peptide analogues.

A Passerini condensation of acyl cyanides, carboxylic acids, and isonitriles has been developed that affords efficient access to functionalized diamides as well as beta-peptides of alpha-hydroxy-beta-amino acids. Such compounds are protease-resistant and form stable helical and sheet structures when incorporated into larger peptides. N-Protected alpha-amino acids and isocyanoesters derived from alpha-amino acids participate in the condensation, leading to alpha/beta peptides embodying the heterogeneous alpha/beta/alpha backbone motif, recent examples of which display antibiotic activity.

New probes for microtubule dynamics.

A phenotype-based screen identifies a purine analog, named diminutol, that perturbs the microtubule cytoskeleton in cells. An affinity-based approach identifies a protein target of this small molecule and leads to the characterization of a new pathway that may regulate cytoskeleton dynamics.

A general approach to medium ring alkynes by using metathesis of cobalt hexacarbonyl containing dienes.

The assembly of medium sized rings (7-9) was achieved by using the metathesis of dienes linked by a cobalt hexacarbonyl complexed alkyne with either Grubbs' or Schrock's catalysts. The products of metathesis were subjected to transformations involving the dicobalt hexacarbonyl complexes, for example, decomplexation to liberate cyclic alkynes or Pauson-Khand reaction.