Comprehensive structure-function analysis reveals gain- and loss-of-function mechanisms impacting oncogenic KRAS activity.

To dissect variant-function relationships in the KRAS oncoprotein, we performed deep mutational scanning (DMS) screens for both wild-type and KRAS mutant alleles. We defined the spectrum of oncogenic potential for nearly all possible variants, identifying several novel transforming alleles and elucidating a model to describe the frequency of mutations in human cancer as a function of transforming potential, mutational probability, and tissue-specific mutational signatures. Biochemical and structural analyses of variants identified in a KRAS second-site suppressor DMS screen revealed that attenuation of oncogenic KRAS can be mediated by protein instability and conformational rigidity, resulting in reduced binding affinity to effector proteins, such as RAF and PI3-kinases, or reduced SOS-mediated nucleotide exchange activity.

BRD-810 is a highly selective MCL1 inhibitor with optimized in vivo clearance and robust efficacy in solid and hematological tumor models.

The MCL1 gene is frequently amplified in cancer and codes for the antiapoptotic protein myeloid cell leukemia 1 (MCL1), which confers resistance to the current standard of care. Therefore, MCL1 is an attractive anticancer target. Here we describe BRD-810 as a potent and selective MCL1 inhibitor and its key design principle of rapid systemic clearance to potentially minimize area under the curve-driven toxicities associated with MCL1 inhibition.

Sotorasib Is a Pan-RASG12C Inhibitor Capable of Driving Clinical Response in NRASG12C Cancers.

Unlabelled: KRASG12C inhibitors, like sotorasib and adagrasib, potently and selectively inhibit KRASG12C through a covalent interaction with the mutant cysteine, driving clinical efficacy in KRASG12C tumors. Because amino acid sequences of the three main RAS isoforms-KRAS, NRAS, and HRAS-are highly similar, we hypothesized that some KRASG12C inhibitors might also target NRASG12C and/or HRASG12C, which are less common but critical oncogenic driver mutations in some tumors. Although some inhibitors, like adagrasib, were highly selective for KRASG12C, others also potently inhibited NRASG12C and/or HRASG12C.

Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site.

Discovery and characterization of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists.

PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo.

Discovery and Structure-Based Design of Potent Covalent PPARγ Inverse-Agonists and .

The ligand-activated nuclear receptor peroxisome-proliferator-activated receptor-γ (PPARG or PPARγ) represents a potential target for a new generation of cancer therapeutics, especially in muscle-invasive luminal bladder cancer where PPARγ is a critical lineage driver. Here we disclose the discovery of a series of chloro-nitro-arene covalent inverse-agonists of PPARγ that exploit a benzoxazole core to improve interactions with corepressors NCOR1 and NCOR2. treatment of sensitive cell lines with these compounds results in the robust regulation of PPARγ target genes and antiproliferative effects.

Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex.

Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes.

Paralog knockout profiling identifies DUSP4 and DUSP6 as a digenic dependence in MAPK pathway-driven cancers.

Although single-gene perturbation screens have revealed a number of new targets, vulnerabilities specific to frequently altered drivers have not been uncovered. An important question is whether the compensatory relationship between functionally redundant genes masks potential therapeutic targets in single-gene perturbation studies. To identify digenic dependencies, we developed a CRISPR paralog targeting library to investigate the viability effects of disrupting 3,284 genes, 5,065 paralog pairs and 815 paralog families.

Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP.

Multimodal small-molecule screening for human prion protein binders.

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including F-observed and saturation transfer difference (STD) NMR spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and screening.

Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor.

Circulating low-density lipoprotein cholesterol (LDLc) is regulated by membrane-bound LDL receptor (LDLr). Upon LDLc and LDLr interaction the complex is internalized by the cell, leading to LDLc degradation and LDLr recycling back to the cell surface. The proprotein convertase subtilisin/kexin type 9 (PCSK9) protein regulates this cycling.

Ligand bioactive conformation plays a critical role in the design of drugs that target the hepatitis C virus NS3 protease.

A ligand-focused strategy employed NMR, X-ray, modeling, and medicinal chemistry to expose the critical role that bioactive conformation played in the design of a variety of drugs that target the HCV protease. The bioactive conformation (bound states) were determined for key inhibitors identified along our drug discovery pathway from the hit to clinical compounds. All adopt similar bioactive conformations for the common core derived from the hit peptide DDIVPC.

Enantiomeric atropisomers inhibit HCV polymerase and/or HIV matrix: characterizing hindered bond rotations and target selectivity.

An anthranilic acid series of allosteric thumb pocket 2 HCV NS5B polymerase inhibitors exhibited hindered rotation along a covalent bond axis, and the existence of atropisomer chirality was confirmed by NMR, HPLC analysis on chiral supports, and computational studies. A thorough understanding of the concerted rotational properties and the influence exerted by substituents involved in this steric phenomenon was attained through biophysical studies on a series of truncated analogues. The racemization half-life of a compound within this series was determined to be 69 min, which was consistent with a class 2 atropisomer (intermediate conformational exchange).

Discovery of novel small-molecule HIV-1 replication inhibitors that stabilize capsid complexes.

The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex.

Discovery of novel P2 substituted 4-biaryl proline inhibitors of hepatitis C virus NS3 serine protease.

Inhibitors of hepatitis C virus NS3 serine protease often incorporate a large P2 moiety to interact with the surface of the enzyme while shielding part of the catalytic triad. This feature is important in many inhibitors in order to have the necessary potency needed for efficacy. In this Letter we explore some new P2 motifs to further exploit this region of the enzyme.

Peptide backbone replacement of hepatitis C virus NS3 serine protease C-terminal cleavage product analogs: discovery of potent succinamide inhibitors.

A number of potent peptidic inhibitors of the NS3 protease have been described in the literature based on a substrate-based approach. In an on-going effort to reduce the peptidic character of this class of inhibitors, two novel series of analogs have been prepared in which the usual P3 amino acid residue is replaced by a succinamide fragment. This new backbone modification not only reduces the peptidic nature of traditional inhibitors but also provides new SAR opportunities for the capping group.

Synthesis and optimization of a novel series of HCV NS3 protease inhibitors: 4-arylproline analogs.

In this report we describe the synthesis and evaluation of diverse 4-arylproline analogs as HCV NS3 protease inhibitors. Introduction of this novel P2 moiety opened up new SAR and, in combination with a synthetic approach providing a versatile handle, allowed for efficient exploitation of this novel series of NS3 protease inhibitors. Multiple structural modifications of the aryl group at the 4-proline, guided by structural analysis, led to the identification of analogs which were very potent in both enzymatic and cell based assays.

A novel inhibitor-binding site on the HIV-1 capsid N-terminal domain leads to improved crystallization via compound-mediated dimerization.

Despite truly impressive achievements in the global battle against HIV there remains a need for new drugs directed against novel targets, and the viral capsid protein (CA) may represent one such target. Intense structural characterization of CA over the last two decades has provided unprecedented insight into the structure and assembly of this key viral protein. Furthermore, several inhibitor-binding sites that elicit antiviral activity have been reported on CA, two of which are located on its N-terminal domain (CANTD).

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 2: structure-activity relationships (SAR) of the C3-phenyl moiety.

Detailed structure-activity relationships of the C3-phenyl moiety that allow for the optimization of antiviral potency of a series of 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione inhibitors of HIV capsid (CA) assembly are described. Combination of favorable substitutions gave additive SAR and allowed for the identification of the most potent compound in the series, analog 27. Productive SAR also transferred to the benzotriazepine and spirobenzodiazepine scaffolds, providing a solution to the labile stereocenter at the C3 position.

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 1: addressing configurational instability through scaffold modification.

The optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly that possess a labile stereocenter at C3 is described. Quaternization of the C3 position of compound 1 in order to prevent racemization gave compound 2, which was inactive in our capsid disassembly assay. A likely explanation for this finding was revealed by in silico analysis predicting a dramatic increase in energy of the bioactive conformation upon quaternization of the C3 position.

Novel inhibitor binding site discovery on HIV-1 capsid N-terminal domain by NMR and X-ray crystallography.

The HIV-1 capsid (CA) protein, a domain of Gag, which participates in formation of both the mature and immature capsid, represents a potential target for anti-viral drug development. Characterization of hits obtained via high-throughput screening of an in vitro capsid assembly assay led to multiple compounds having this potential. We previously presented the characterization of two inhibitor series that bind the N-terminal domain of the capsid (CA(NTD)), at a site located at the bottom of its helical bundle, often referred to as the CAP-1 binding site.

Monitoring binding of HIV-1 capsid assembly inhibitors using (19)F ligand-and (15)N protein-based NMR and X-ray crystallography: early hit validation of a benzodiazepine series.

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)-1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high-throughput screening using an in vitro capsid assembly assay (CAA).

Molecular mechanism by which a potent hepatitis C virus NS3-NS4A protease inhibitor overcomes emergence of resistance.

Although optimizing the resistance profile of an inhibitor can be challenging, it is potentially important for improving the long term effectiveness of antiviral therapy. This work describes our rational approach toward the identification of a macrocyclic acylsulfonamide that is a potent inhibitor of the NS3-NS4A proteases of all hepatitis C virus genotypes and of a panel of genotype 1-resistant variants. The enhanced potency of this compound versus variants D168V and R155K facilitated x-ray determination of the inhibitor-variant complexes.

Inhibition of HIV-1 capsid assembly: optimization of the antiviral potency by site selective modifications at N1, C2 and C16 of a 5-(5-furan-2-yl-pyrazol-1-yl)-1H-benzimidazole scaffold.

A uHTS campaign led to the discovery of a 5-(5-furan-2-ylpyrazol-1-yl)-1H-benzimidazole series that inhibits assembly of HIV-1 capsid. Synthetic manipulations at N1, C2 and C16 positions improved the antiviral potency by a . The X-ray structure of 33 complexed with the capsid N-terminal domain allowed identification of major interactions between the inhibitor and the protein.