Disulfide tethering is a site-directed method of drug discovery used to identify hits for challenging targets. We applied tethering to target oncogenic KRAS, a small GTPase once considered undruggable due to its high nucleotide affinity and a perceived absence of binding sites. We prepared a library of 2160 disulfide-containing fragments. We screened over 1000 compounds against a panel of 83 engineered cysteine mutants of KRAS G12D in the active conformation and screened the full library for a subset of 30 mutants. For select mutants and hits, we performed 2-mercaptoethanol competition assays (βME-50) to prioritize ligands. Ligandability analysis comparing hit rates across mutant residues enabled the identification of druggable hot spots. Our studies confirmed known binding sites, including the Switch-II / α-helix 3 pocket. In addition, we identified previously undescribed cryptic pockets and validated select hits using computational chemistry and NMR spectroscopy. These pockets represent promising opportunities for future drug discovery campaigns.

Abstract KRAS oncogenic mutations, including the common G12C and G12D variants, impair GTP hydrolysis and occur at high frequency in cancer. The unexplained variations in RAS mutant allele frequency and tissue distribution, and the question of whether mutant-specific preferences for effector binding contribute to their prevalence in cancer, remain unanswered. Our NMR solution structures reveal that the SW2-pocket (SIIP) is larger in KRASG12D than in KRASG12C. This difference arises from distinct conformations of the P-loop, the SW2 loop, and the N-terminal a2 helix that are coordinated by perturbations in SW1 region. Codon 12 in the P-loop and residue Q61 within SW2 are key contributors to these structural differences. KRASG12D shows multiple D12 rotamers, whereas KRASG12C adopts a single C12 rotamer that restricts access to the nucleotide gamma-phosphate (gP) which imposes constraints on SIIP-directed inhibitor design. The Q61 side chain also adopts mutant-specific packing: in KRASG12C the side chain is constrained by interaction with Y96 leading to a narrowed pocket compared to that in KRASG12D where it shifts away from the SIIP, increasing its distance from the gP. These distinctions suggest that the mutants alter the catalytic environment for GTP hydrolysis. Our analysis of protein dynamics by NMR supports unique SW2 packing observed in KRASG12D. We identify a potent pan-KRAS small-molecule inhibitor, BBO-11534, that binds both GTP- and GDP-loaded KRAS. 31P NMR analyses demonstrate that BBO-11534 shifts the conformational equilibrium of GTP-bound KRASWT and KRASG12D toward the signaling-incompetent state 1. In contrast, this shift is modest in GTP-KRASG12C, likely due to confinement of the restricted C12 rotamer and smaller SIIP, which limits inhibitor access to the gP. This distinct conformational network within the active site provides mechanistic rationale for these allele-specific responses. To assess the impact of these structural differences between KRASG12D vs KRASG12C, we examined PI3K p110α binding in cells using a Bioluminescence Resonance Energy Transfer (BRET) assay. These measurements show that KRASG12D binds p110α with the highest affinity, followed by KRASG12C and then KRASWT. Chemical shift perturbation NMR and molecular dynamics simulations recapitulate this binding hierarchy (G12D > G12C > WT). Likewise, immunoprecipitation of FLAG-tagged KRASG12D in HEK293T cells captures more PI3Kα than the KRASG12C or KRASWT proteins, further supporting this trend. Together, this study identifies unique structural features in oncogenic KRAS mutants that demonstrate how these features influence protein function and provide guidance for the design of allele-specific therapeutics. Citation Format: ALOK K. SHARMA, Megan Rigby, MARCO TONELLI, Nicole FER, Patrick Alexander, JUN PEI, YUE YANG, DANA Rabara, Erik K. Larsen, Brian P. Smith, MA ROGER, Vandana Kumari, Marcin Dyba, Felice Lightstone, BIN WANG, PEDRO J. BELTRAN, Eli Wallace, Andrew G. Stephen, Dwight V. Nissley, Frank McCormick, Anna E. Maciag. KRAS G12C and G12D mutants exhibit distinct conformational flexibility in the helix 3–switch 2 pocket that drives differential protein function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr A047.

RAS is the most frequently mutated oncogene in cancer. RAS proteins show high sequence similarities in their G-domains but are significantly different in their C-terminal hypervariable regions (HVR). These regions interact with the cell membrane via lipid anchors that result from posttranslational modifications (PTM) of cysteine residues. KRAS4b is unique as it has only one cysteine that undergoes PTM, C185. Small molecule covalent modification of C185 would block any form of prenylation and subsequently inhibit attachment of KRAS4b to the cell membrane, blocking its biological activity. We translated this concept to the discovery and development of disulfide tethering screen hits into irreversible covalent modifiers of C185. These compounds inhibited proliferation of KRAS4b-driven mouse embryonic fibroblasts, but not cells driven by N-myristoylated KRAS4b that harbor a C185S mutation and are not dependent on C185 prenylation. Top–down proteomics was used to confirm target engagement in cells. These compounds bind in a pocket formed when the HVR folds back between helix 3 and 4 in the G-domain (HVR-α3-α4). This interaction can happen in the absence of small molecules as predicted by molecular dynamics simulations and is stabilized in the presence of C185 binders as confirmed by small-angle X-ray scattering and solution NMR. NOESY-HSQC, an NMR approach that measures internuclear distances of 6 Å or less, and structure analysis identified the critical residues and interactions that define the HVR-α3-α4 pocket. Further development of compounds that bind to this pocket could be the basis of a new approach to targeting KRAS cancers.

Up to 30% of cancers harbor activating mutations in one of three RAS genes, HRAS, NRAS, or KRAS, that drive uncontrolled cellular proliferation, alter cellular metabolism, movement, differentiation, and promote cell survival and tumor immune evasion. The frequency of mutation varies across alleles and specific substitutions, with KRAS mutations occurring in roughly 81% of patients, and KRAS G12D, KRAS G12V, and KRAS G12C substitutions constituting 33%, 23%, and 12% of these cases, respectively. Cancer types exhibit characteristic preferences for RAS substitutions, the reason for which is still unknown. Oncogenic amino acid substitutions have been found to endow subtle differences in biochemical properties which cannot be explained by existing X-ray structural models. In addition, it is unclear whether these translate to distinctions in downstream signaling potential. We have initiated this study with the overall goal of understanding the effect of oncogenic mutations on the structure and dynamic properties of KRAS4b proteins in solution, and to identify any ensuing differences in effector binding behavior. Here, we present the data that identifies the structural differences between the common mutants KRAS G12D and KRAS G12C and the wildtype protein in the active conformation using solution-state NMR and Molecular Dynamics (MD) simulations, that could likely be coupled with the differing capacities to engage downstream effectors. Chemical shift perturbation (CSP) analysis demonstrates that oncogenic mutations in codon 12 of KRAS4b induce significant conformational differences when compared to the wildtype protein. These observations are localized to the Switch 2 region. We validate these observations in three-dimensional structures and reveal critical differences that are conferred by these oncogenic mutants. The solved three-dimensional solution structures of KRAS G12C/T35S/C118S-GppNHp and KRAS G12D/T35S/C118S-GppNHp reveal remarkable conformational differences within the Switch II regions, with the G12D-mutant protein adopting a relatively more flexible Switch II conformation than the G12C-mutant. Our MD simulations at a 2 µs timescale support these observations. Furthermore, we observed downstream signaling differences between KRAS G12C and KRAS G12D mutants in several cell line systems. If observed structural differences between KRAS mutants are found to directly translate to effector binding affinity, it would provide critical insight into why cancer types display preferences for RAS substitutions, and would inform RAS-targeted therapeutics. Citation Format: Brian Smith, Alok K. Sharma, Roger Ma, Megan Rigby, Vandana Kumari, Dominic Esposito, Dwight Nissley, Frank McCormick, Anna E. Maciag. KRAS codon 12 oncogenic mutations modulate protein conformation within the Switch II/Helix3 pocket [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A033.

Cell growth and division are coordinated, ensuring homeostasis under any given growth condition, with division occurring as cell mass doubles. The signals and controlling circuit(s) between growth and division are not well understood; however, it is known in Escherichia coli that the essential GTPase Era, which is growth rate regulated, coordinates the two functions and may be a checkpoint regulator of both. We have isolated a mutant of Era that separates its effect on growth and division. When overproduced, the mutant protein Era647 is dominant to wildtype Era and blocks division, causing cells to filament. Multicopy suppressors that prevent the filamentation phenotype of Era647 either increase the expression of FtsZ or decrease the expression of the Era647 protein. Excess Era647 induces complete delocalization of Z rings, providing an explanation for why Era647 induces filamentation, but this effect is probably not due to direct interaction between Era647 and FtsZ. The hypermorphic ftsZ* allele at the native locus can suppress the effects of Era647 overproduction, indicating that extra FtsZ is not required for the suppression, but another hypermorphic allele that accelerates cell division through periplasmic signaling, ftsL*, cannot. Together, these results suggest that Era647 blocks cell division by destabilizing the Z ring. IMPORTANCE All cells need to coordinate their growth and division, and small GTPases that are conserved throughout life play a key role in this regulation. One of these, Era, provides an essential function in the assembly of the 30S ribosomal subunit in Escherichia coli, but its role in regulating E. coli cell division is much less well understood. Here, we characterize a novel dominant negative mutant of Era (Era647) that uncouples these two activities when overproduced; it inhibits cell division by disrupting assembly of the Z ring, without significantly affecting ribosome production. The unique properties of this mutant should help to elucidate how Era regulates cell division and coordinates this process with ribosome biogenesis.

Abstract Tipifarnib, a drug that targets HRAS through inhibiting farnesyl transferase, is in phase II clinical trials and appears to show activity in tumors harboring oncogenic HRAS mutations (https://kuraoncology.com/pipeline/#tipifarnib). Unfortunately, these drugs are not expected to be effective on KRAS cancers because KRAS, unlike HRAS, can be prenylated by geranylgeranyl transferase following farnesyl transferase inhibition. To address this, we have developed compounds that prevent farnesylation of KRAS 4B by covalent reaction with C185, the site of prenylation. These compounds bind to a pocket in the G-domain that is formed by interaction with the hypervariable region, an interaction that does not seem to occur in other RAS proteins. This existence of this pocket has been demonstrated through biochemical and biophysical analysis, including NMR and small-angle X-ray scattering. The compounds we have developed are active in cells: they prevent proliferation of MEFs driven by oncogenic KRAS proteins but do not affect MEFs supported by myristoylated KRAS G12D C185S at equivalent concentrations. We are currently optimizing these compounds for further preclinical development. Citation Format: Anna Maciag, Yue Yang, David Turner, Marcin Dyba, Vandana Kumari, Brian Smith, Lixin Fan, Stephan Gysin, Andrew Wolfe, Hazem Abdelkarim, Vadim Gaponenko, Felice Lightstone, Dwight Nissley, Frank McCormick. Preventing KRAS processing [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA21.

Dietary isothiocyanates abundant as glucosinolate precursors in many edible cruciferous vegetables are effective for prevention of cancer in chemically-induced and transgenic rodent models. Some of these agents, including phenethyl isothiocyanate (PEITC), have already advanced to clinical investigations. The primary route of isothiocyanate metabolism is its conjugation with glutathione (GSH), a reaction catalyzed by glutathione S-transferase (GST). The pi class GST of subunit type 1 (hGSTP1) is much more effective than the alpha class GST of subunit type 1 (hGSTA1) in catalyzing the conjugation. Here, we report the crystal structures of hGSTP1 and hGSTA1 each in complex with the GSH adduct of PEITC. We find that PEITC also covalently modifies the cysteine side chains of GST, which irreversibly inhibits enzymatic activity.

Overexpressed glutathione S-transferase P1 (GSTP1) has been associated with chemotherapy resistance. GSTP1 activated arylated diazeniumdiolates causes significant DNA damage by nitric oxide (NO) release. Poly(ADP-ribose) polymerases (PARP) inhibitors induces DNA damage. A hybrid prodrug was designed by combining the structural features of established PARP-1 inhibitors and arylated diazeniumdiolates to exacerbate DNA damage. The prodrug is activated by GSTP1 catalyzed reaction with glutathione and releases NO and a moiety that inhibits PARP-1. It has also proven to be effective as anticancer agent in both in vitro and in vivo models. The chemopotentiating effects of the prodrug were also observed in combination with microtubule targeting agents or proteasome inhibitors, accompanied by increase in DNA damage, and induction of apoptosis. Interestingly, S-nitrosation of Cys101 of GSTP1 by the prodrug upon activation by GSTP1 was observed. This modification significantly decreased the catalytic activity of GSTP1, suggesting that the prodrug executes synergistic effects through multiple cellular effects, including direct inhibitory effect on GSTP1. These findings demonstrate that S-nitrosation of GSTP1 may affect proper protein folding and induce unfolded protein and endoplasmic reticulum stress. Our strategy not only activates prodrug by GSTP1 catalytic activity but also inhibits GSTP1 upon activation and delivers the compound at the cancer site. Thus, a three-pronged attack on cancer cells may be afforded by such a novel strategy. Additionally, GSTP1 inhibition by designed prodrug may provide a translational opportunity to target GSTP1 for its role in MAP kinase cascades, which participates in cellular survival and death signaling. As future studies are warranted to confirm our hypothesis, we plan to continue our structure based design to improve the hybrid prodrug for higher GSTP1 selectivity and higher bioavailability.

Abstract We have shown previously that withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from an Ayurvedic medicine plant (Withania somnifera), not only inhibits growth of human breast cancer cells in vitro and in vivo but also prevents mammary cancer development in a transgenic mouse model without any toxicity. However, the mechanisms underlying cancer preventive effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA in human breast cancer cells. WA treatment resulted in G2 and mitotic arrest in MCF-7 (estrogen-receptor positive), SK-BR-3 (estrogen-receptor negative), and SUM159 (triple-negative) cells in association with a marked decrease in protein level of β-tubulin. These effects were not observed with the naturally-occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). In addition, a non-tumorigenic normal human mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest and tubulin downregulation by WA treatment compared with breast cancer cells. Downregulation of tubulin protein in WA-treated cells was due to transcriptional repression as well as proteasomal degradation. Vehicle-treated control breast cancer cells exhibited a normal bi-polar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of the normal spindle morphology in breast cancer cells. Unlike breast cancer cells, WA-treated MCF-10A cells exhibited aggregation of tubulin around nucleus. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA at cysteine-303 of β-tubulin in MCF-7 cells. Molecular docking studies indicated that the WA-binding pocket was located on the surface of the β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of anticancer effect of WA. This work was supported by the grants RO1 CA142604 and P30 CA047904 awarded by the National Cancer Institute. Citation Format: Shivendra V. Singh, Marie Lue Antony, Joomin Lee, Eun-Ryeong Hahm, Su-Hyeong Kim, Guillermo Romero, Adam I. Marcus, Zhen Yang, Vandana Kumari, Xinhua Ji, Courtney L. Vowell, Peter Wipf, Guy T. Uechi, Nathan A. Yates. Withaferin A downregulates tubulins and covalently binds β-tubulin at cysteine-303 in human breast cancer cells. [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 228. doi:10.1158/1538-7445.AM2014-228

We report the antitumor effects of nitric oxide (NO) releasing derivatives of the PARP-1 inhibitor olaparib (1). Compound 5b was prepared by coupling the carboxyl group of 3b and the free amino group of arylated diazeniumdiolated piperazine 4. Analogue 5a has the same structure except that the F is replaced by H. Compound 13 is the same as 5b except that a Me2N-N(O)═NO- group was added para and ortho to the nitro groups of the dinitrophenyl ring. The resulting prodrugs are activated by glutathione in a reaction accelerated by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancers. This metabolism generates NO plus a PARP-1 inhibitor simultaneously, consuming reducing equivalents, leading to DNA damage concomitant with inhibition of DNA repair, and in the case of 13 inducing cross-linking glutathionylation of proteins. Compounds 5b and 13 reduced the growth rates of A549 human lung adenocarcinoma xenografts with no evidence of systemic toxicity.

The IL-6/GP130/STAT3 pathway is critical for the progression of multiple types of cancers. We report here the discovery of raloxifene and bazedoxifene as novel inhibitors of IL-6/GP130 protein-protein interactions (PPIs) using multiple ligand simultaneous docking (MLSD) and drug repositioning approaches. Multiple drug scaffolds were simultaneously docked into hot spots of GP130 D1 domain by MLSD to compete with the key interacting residues of IL-6, followed by tethering to generate virtual hit compounds. Similarity searches of virtual hits on drug databases identified raloxifene and bazedoxifene as potential inhibitors of IL-6/GP130 interaction. In cancer cell assays both compounds bind to GP130 and demonstrated selective inhibition of IL-6 induced STAT3 phosphorylation and were significantly more potent than the previously reported natural product inhibitor MDL-A. The identified drugs represent a new class of lead compounds with piperidine, benzothiophene, and indole scaffolds to inhibit IL-6 induced homodimerization of GP130. Besides potential direct usage for clinic trials, the two compounds can also serve as lead compounds for optimization to speed the development of drugs selectively targeting the IL-6/GP130/STAT3 cancer signaling pathway.

Background: The tubulin microtubule network remains an attractive anticancer target. Results: The antitumor steroidal lactone withaferin A (WA) down-regulates tubulin and binds to Cys303 of β-tubulin. Conclusion: Tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. Significance: Favorable safety and pharmacokinetic profiles merit clinical investigation of WA for prevention and/or treatment of breast cancer. Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of β-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys303 of β-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.