Covalent Small Molecule Research Articles

Correlation of the in vitro biotransformation of H3B-6527 in dog and human hepatocytes with the in vivo metabolic profile of 14C-H3B-6527 in a dog mass balance study

1. H3B-6527 is an orally available covalent small molecule inhibitor of FGFR4 undergoing evaluation in adults with hepatocellular carcinoma. Absorption, metabolism, transport and elimination of H3B-6527 were investigated in vitro and in a 14C-H3B-6527 beagle dog mass balance study.2. Following intravenous dosing in dogs, unchanged 14C-H3B-6527 represents only 1.6% of the total dose in excreta. The low amount of radioactivity in the dog urine (4.9% of the administered dose), suggests that renal elimination is a minor pathway of clearance for H3B-6527. A majority of the radioactivity was observed in the feces up to 5 days after dose administration, suggesting that drug-related material was secreted in the bile, and that H3B-6527 clearance was mostly driven by metabolism.3. In vitro, H3B-6527 is a substrate of GSTs, CYP3A and P-glycoprotein.4. The major pathways of metabolism were similar in human and dog hepatocytes, and occurred via glutathione (GSH) conjugations and sequential hydrolysis, N-deethylation and hydroxylation.5. The metabolic profile of H3B-6527 was qualitatively similar in dog hepatocytes and plasma/excreta.Trial registration: ClinicalTrials.gov identifier: NCT02834780.

Abstract 4484: Discovery and in vitro characterization of AMG 510–a potent and selective covalent small-molecule inhibitor of KRASG12C

Abstract Activating mutations in RAS represent the most common oncogenic driver mutation in cancer. The single amino acid substitution of cysteine for glycine at position 12 (KRASG12C) is frequently found in solid malignancies, particularly in lung adenocarcinoma (~13%), colorectal adenocarcinoma (3%), and pancreatic adenocarcinoma (~1%). Recently it has been demonstrated that KRASG12C can be targeted with covalent small molecule inhibitors which react with the mutant cysteine adjacent to the switch II pocket (SIIP), locking KRAS in its inactive GDP-bound state. We describe here the discovery and in vitro characterization of AMG 510, a covalent inhibitor of KRASG12C possessing potent biochemical and cellular activity, as well as robust in vivo efficacy. AMG 510 inhibited SOS1-catalyzed nucleotide exchange of recombinant mutant KRASG12C/C118A but had minimal effect on KRASC118A, which is wildtype at position 12. The observed rate constant (kinact/Ki) of covalent modification of KRASG12C by AMG 510 was determined biochemically by mass spectrometry as well as in the cellular context (kobs/[I]). Cysteine proteome analysis of cells treated with AMG 510 revealed that only the G12C-containing peptide of KRAS was covalently modified. AMG 510 inhibited KRAS signaling as measured by ERK phosphorylation in all KRAS p.G12C cell lines tested, but did not inhibit phosphorylation of ERK in cell lines lacking the KRAS p.G12C mutation. Cellular occupancy of KRASG12C by AMG 510 was determined by mass spectrometry and correlated well with inhibition of ERK phosphorylation. AMG 510 also selectively impaired the viability of KRAS p.G12C mutant lines. Combination treatment of AMG 510 with inhibitors of other cellular signaling pathways exhibited evidence for synergistic effects on cell viability. Treatment of KRAS p.G12C lines with covalent KRASG12C inhibitors increased the expression of HLA. To test the impact of KRASG12C inhibition on immune surveillance in vivo, we generated a syngeneic tumor cell line that is suitable for testing AMG 510 in combination with checkpoint inhibitor therapies and characterized this line in vitro. AMG 510 is currently being evaluated in a Phase I study in patients with solid tumors harboring KRAS p.G12Cmutations. Citation Format: Anne Y. Saiki, Kevin Gaida, Karen Rex, Pragathi Achanta, Tisha San Miguel, Neelima Koppada, Dhanashri Bagal, Brian A. Lanman, Robert S. Foti, John D. McCarter, Laurie P. Volak, Jude Canon, Victor J. Cee, J. Russell Lipford. Discovery and in vitro characterization of AMG 510–a potent and selective covalent small-molecule inhibitor of KRASG12C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4484.

Covalent Plasmodium falciparum-selective proteasome inhibitors exhibit a low propensity for generating resistance in vitro and synergize with multiple antimalarial agents.

Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the β2, β5 or β6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only <five-fold decreases in parasite susceptibility. This result compares favorably against previously published non-covalent inhibitors of the Plasmodium proteasome that can select for resistant parasites with >hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria.

The Alkyne Moiety as a Latent Electrophile in Irreversible Covalent Small Molecule Inhibitors of Cathepsin K.

Irreversible covalent inhibitors can have a beneficial pharmacokinetic/pharmacodynamics profile but are still often avoided due to the risk of indiscriminate covalent reactivity and the resulting adverse effects. To overcome this potential liability, we introduced an alkyne moiety as a latent electrophile into small molecule inhibitors of cathepsin K (CatK). Alkyne-based inhibitors do not show indiscriminate thiol reactivity but potently inhibit CatK protease activity by formation of an irreversible covalent bond with the catalytic cysteine residue, confirmed by crystal structure analysis. The rate of covalent bond formation (kinact) does not correlate with electrophilicity of the alkyne moiety, indicative of a proximity-driven reactivity. Inhibition of CatK-mediated bone resorption is validated in human osteoclasts. Together, this work illustrates the potential of alkynes as latent electrophiles in small molecule inhibitors, enabling the development of irreversible covalent inhibitors with an improved safety profile.

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.

Abstract 2796: Predicting in vivo efficacy from in vitro data: Quantitative systems pharmacology modeling for an epigenetic modifier drug in cancer

Abstract Epigenetic modulators are increasing in prominence as potential cancer therapies. These drugs achieve their effectiveness by inducing transcriptional changes that can inhibit cancer progression. Here, we focus on a potent and selective covalent small molecule inhibitor of LSD1 (RO7051750/ORY-1001), a lysine-specific histone demethylase enzyme. LSD1 inhibition causes epigenetic reprogramming of cancer cells by downregulating pro-proliferative neuroendocrine genes, inducing a pro-differentiation, cytostatic effect. Furthermore, the durability of this response is enhanced by covalent drug binding and the resulting dependence on target turnover. To explore the impact of dose and scheduling on the pharmacodynamic profiles underlying treatment efficacy, we developed a predictive model of drug effect, which incorporates experimentally-derived pharmacokinetic and pharmacodynamic data. This mechanistic mathematical model was calibrated based on in vitro cell-type specific kinetic data, which possesses high dimensionality across time points and dose ranges. The model describes drug-induced target engagement of LSD1, which acts as a transcriptional regulator of neuroendocrine gene, GRP. GRP levels drive reversible epigenetic “switching” between a proliferating (denoted P) and a quiescent (denoted Q) cell population (low GRP: P to Q; high GRP: Q to P), capturing the cytostatic effects of the drug. The model was trained with in vitro measurements of target engagement, GRP mRNA levels, and cell growth inhibition (including drug-free cell growth) across various time points and dosages from a small cell lung cancer cell line (NCI-H510A). Importantly, GRP mRNA and cell viability dynamics were also captured upon drug withdrawal, which enables the modeling of drug effect durability. The resulting model quantitatively describes the relationship between drug dose, target engagement, biomarker and cell growth dynamics in vitro. To scale to the in vivo setting, we integrated the in vitro-trained pharmacodynamic model with a pharmacokinetic model describing drug distribution and clearance in mouse. This hybrid model accurately predicted in vivo tumor growth dynamics in the NCI-H510A mouse xenograft across a range of doses and schedules. We used the model to explore the target engagement and biomarker profiles underlying treatment efficacy, both in vitro and in vivo. In conclusion, by training our model on key cell-type specific in vitro kinetic data we were able to accurately predict anti-tumor growth effects in vivo. This quantitative modeling framework highlights the biological profiles underlying efficacy for this latest generation of target-selective epigenetic modifying drugs. Citation Format: Mehdi Bouhaddou, Li J. Yu, Serena Lunardi, Spyros K. Stamatelos, Fiona Mack, James M. Gallo, Marc R. Birtwistle, Antje-Christine Walz. Predicting in vivo efficacy from in vitro data: Quantitative systems pharmacology modeling for an epigenetic modifier drug in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2796.

Identification of Noncatalytic Lysine Residues from Allosteric Circuits via Covalent Probes.

Covalent modifications of nonactive site lysine residues by small molecule probes has recently evolved into an important strategy for interrogating biological systems. Here, we report the discovery of a class of bioreactive compounds that covalently modify lysine residues in DegS, the rate limiting protease of the essential bacterial outer membrane stress response pathway. These modifications lead to an allosteric activation and allow the identification of novel residues involved in the allosteric activation circuit. These findings were validated by structural analyses via X-ray crystallography and cell-based reporter systems. We anticipate that our findings are not only relevant for a deeper understanding of the structural basis of allosteric activation in DegS and other HtrA serine proteases but also pinpoint an alternative use of covalent small molecules for probing essential biochemical mechanisms.

Potent and Selective Covalent Quinazoline Inhibitors of KRAS G12C

Targeted covalent small molecules have shown promise for cancers driven by KRAS G12C. Allosteric compounds that access an inducible pocket formed by movement of a dynamic structural element in KRAS, switch II, have been reported, but these compounds require further optimization to enable their advancement into clinical development. We demonstrate that covalent quinazoline-based switch II pocket (SIIP) compounds effectively suppress GTP loading of KRAS G12C, MAPK phosphorylation, and the growth of cancer cells harboring G12C. Notably we find that adding an amide substituent to the quinazoline scaffold allows additional interactions with KRAS G12C, and remarkably increases the labeling efficiency, potency, and selectivity of KRAS G12C inhibitors. Structural studies using X-ray crystallography reveal a new conformation of SIIP and key interactions made by substituents located at the quinazoline 2-, 4-, and 7-positions. Optimized lead compounds in the quinazoline series selectively inhibit KRAS G12C-dependent signaling and cancer cell growth at sub-micromolar concentrations.

2-Chloropropionamide As a Low-Reactivity Electrophile for Irreversible Small-Molecule Probe Identification.

Resurgent interest in covalent target engagement in drug discovery has demonstrated that small molecules containing weakly reactive electrophiles can be safe and effective therapies. Several recently FDA-approved drugs feature an acrylamide functionality to selectively engage cysteine side chains of kinases (Ibrutinib, Afatinib, and Neratinib). Additional electrophilic functionalities whose reactivity is compatible with highly selective target engagement and in vivo application could open new avenues in covalent small molecule discovery. Here, we report the synthesis and evaluation of a library of small molecules containing the 2-chloropropionamide functionality, which we demonstrate is less reactive than typical acrylamide electrophiles. Although many library members do not appear to label proteins in cells, we identified S-CW3554 as selectively labeling protein disulfide isomerase and inhibiting its enzymatic activity. Subsequent profiling of the library against five diverse cancer cell lines showed unique cytotoxicity for S-CW3554 in cells derived from multiple myeloma, a cancer recently reported to be sensitive to PDI inhibition. Our novel PDI inhibitor highlights the potential of 2-chloropropionamides as weak and stereochemically tunable electrophiles for covalent drug discovery.

Targeting NF-κB p65 with a Helenalin Inspired Bis-electrophile.

The canonical NF-κB signaling pathway is a mediator of the cellular inflammatory response and a target for developing therapeutics for multiple human diseases. The furthest downstream proteins in the pathway, the p50/p65 transcription factor heterodimer, have been recalcitrant toward small molecule inhibition despite the substantial number of compounds known to inhibit upstream proteins in the activation pathway. Given the roles of many of these upstream proteins in multiple biochemical pathways, targeting the p50/p65 heterodimer offers an opportunity for enhanced on-target specificity. Toward this end, the p65 protein presents two nondisulfide cysteines, Cys38 and Cys120, at its DNA-binding interface that are amenable to targeting by covalent molecules. The natural product helenalin, a sesquiterpene lactone, has been previously shown to target Cys38 on p65 and ablate its DNA-binding ability. Using helenalin as inspiration, simplified helenalin analogues were designed, synthesized, and shown to inhibit induced canonical NF-κB signaling in cell culture. Moreover, two simplified helenalin probes were proficient at forming covalent protein adducts, binding to Cys38 on recombinant p65, and targeting p65 in HeLa cells without engaging canonical NF-κB signaling proteins IκBα, p50, and IKKα/β. These studies further support that targeting the p65 transcription factor-DNA interface with covalent small molecule inhibitors is a viable approach toward regulating canonical NF-κB signaling.

Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA.

One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)(exp)) present in a 5' untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)(exp) in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)(exp), which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides.

Abstract 750: JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative breast cancer

Abstract Background: Patients with triple-negative breast cancer (TNBC) have a very poor prognosis due to metastasis and limited targeted therapies. Basal-like TNBC has hyperactivated JNK (c-Jun N-terminal kinase). JNK plays a vital role in malignant transformation in different cancers and self-renewal and maintenance of glioma cancer stem cells (CSCs). However, it is not known whether JNK signaling is a clinically relevant target in TNBC. We tested the hypotheses that JNK signaling plays a fundamental role in TNBC tumorigenesis and metastasis by promoting CSCs and that suppressing JNK signaling using JNK-IN-8 inhibits tumorigenicity and metastasis of TNBC cells. Methods: We examined the effects of JNK knockdown or overexpression on cellular functions of TNBC cells and CSC subpopulations in vitro. We also evaluated the anti-TNBC efficacy of JNK-IN-8, an ATP-directed covalent small molecule inhibitor of JNK that specifically binds the Cys116 on JNK. Results: In patients with TNBC (n=79), low c-Jun mRNA level was associated with better disease-free survival (DFS) (P=0.025), and there was a tendency (P=0.167) toward better DFS with low JNK1 mRNA level. No similar trend was observed for JNK2. These results suggest that JNK1 and c-Jun may have clinical significance in TNBC. Knockdown of JNK1 or JNK2 led to reductions in mammosphere formation, migration, and invasion in HCC70 and SUM149 cells. In contrast, overexpression of constitutively active MKK7-JNK1 or -JNK2 enhanced these cellular activities and increased the ALDH1+ subpopulation. These data indicate a potential role of JNK signaling in regulating TNBC tumorigenesis and metastasis, possibly by promoting CSCs. JNK-IN-8 suppressed proliferation, anchorage-independent growth, migration, and invasion in HCC70 and SUM149 cells. In addition, JNK-IN-8 also reduced mammosphere formation and the CD44+/CD24- and ALDH1+ subpopulations, suggesting that JNK-IN-8 can inhibit JNK signaling-mediated CSC self-renewal and maintenance. Furthermore, overexpression of mutant JNK1- or JNK2-C116S, in which Cys116 was replaced with Ser116, partially abolished the inhibitory effects of JNK-IN-8 on cellular functions, suggesting that JNK-IN-8 executes antitumor activity by specifically targeting JNK. Lastly, JNK inhibition by JNK-IN-8 led to down-regulation of Notch1, suggesting involvement of Notch1 in JNK signaling-mediated CSC regulation. Conclusion: JNK signaling regulates TNBC tumorigenesis and metastasis by promoting CSCs partly via Notch signaling. Our data demonstrate that JNK is an important therapeutic target in TNBC, providing a rationale for further preclinical and clinical development of a JNK-targeted therapy for TNBC. JNK-IN-8 is an effective inhibitor targeting JNK signaling in TNBC. Our studies justify searching for JNK isoform selective inhibitors since there is evidence that JNK1 and JNK2 can have opposing roles in certain settings. Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupunganti, Tinghu Zhang, Takahiro Kogawa, Gaurav B. Chauhan, Nathanael S. Gray, Chandra Bartholomeusz, Kevin N. Dalby, Naoto T. Ueno. JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 750. doi:10.1158/1538-7445.AM2014-750

Dissecting allosteric effects of activator-coactivator complexes using a covalent small molecule ligand.

Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in koff. Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both kon and koff, suggesting stabilization of the binary complex.

Tuning probe selectivity for chemical proteomics applications

Covalent chemical probes enable investigation of a desired fraction of the proteome. It is possible to adjust the selectivity of these probes, so they either react with a certain amino acid in all proteins, a class of proteins or only a single protein species. A combination of specific reactive groups with additional recognition elements can fine tune probes to hit the desired proteins, even in the presence of related family members. Using probes of lower or higher selectivity, screening experiments for inhibitor discovery and imaging experiments for localization studies can be performed, showing only a fraction of the power of covalent small molecule probes.

Letting go of inhibition

UCSF researchers have created a class of reversible, covalent small molecule kinase inhibitors that could be more selective and safer than previous kinase inhibitors. Principia Biopharma has exclusively licensed the technology and hopes to develop new compounds to treat cancer and autoimmune diseases.

Rational Design of Inhibitors and Activity-Based Probes Targeting Clostridium difficile Virulence Factor TcdB

Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs.