Bioorganic & Medicinal Chemistry Letters

Substance use and related mental health epidemics are causing increasing suffering and death in diverse communities.1,2 Despite extensive efforts focused on developing pharmacotherapies for treating substance use disorders, there is an urgent need for radically different therapeutic approaches.3,4 Ibogaine provides an important drug prototype in this direction, as a psychoactive iboga alkaloid suggested to have the ability to interrupt opioid use in drug-dependent humans.5 However, ibogaine and its major metabolite noribogaine present considerable safety risk associated with cardiac arrhythmias.6 We introduce a new class of iboga alkaloids - "oxa-iboga" - defined as benzofuran-containing iboga analogs and created via structural editing of the iboga skeleton. The oxa-iboga compounds act as potent kappa opioid receptor agonists in vitro and in vivo, but exhibit atypical behavioral features compared to standard kappa psychedelics. We show that oxa-noribogaine has greater therapeutic efficacy in rat models of opioid use, and no cardiac pro-arrhythmic potential, in contrast to noribogaine. Oxa-noribogaine induces long-lasting suppression of morphine and fentanyl intake after a single dose, persistent reduction of morphine intake and reinforcing efficacy after a short treatment regimen, and suppression of morphine and fentanyl drug seeking in relapse models. Oxa-noribogaine also induces a lasting elevation of neurotrophin proteins in the ventral tegmental area and medial prefrontal cortex, consistent with targeted neuroplasticity induction and alteration of addiction-like states. As such, oxa-iboga compounds represent candidates for a novel type of pharmacotherapy for treatment of opioid use disorder.

Despite their widespread impact on human health there are no approved drugs for combating alphavirus infections. The heterocyclic {beta}-aminomethyl vinyl sulfone RA-0002034 (1a) is a potent irreversible covalent inhibitor of the alphavirus nsP2 cysteine protease with broad spectrum antiviral activity. Analogs of 1a that varied each of three regions of the molecule were synthesized to establish structure-activity relationships for inhibition of Chikungunya (CHIKV) nsP2 protease and viral replication. The covalent warhead was highly sensitive to modifications of the sulfone or vinyl substituents. However, numerous alterations to the core 5-membered heterocycle and its aryl substituent were well tolerated and several analogs were identified that enhanced CHIKV nsP2 binding. For example, the 4-cyanopyrazole analog 8d exhibited a kinact/Ki ratio >10,000 M-1s-1. 3-Arylisoxazole was identified an isosteric replacement for the 5-membered heterocycle, which circumvented the intramolecular cyclization that complicated the synthesis of pyrazole-based inhibitors like 1a. The accumulated structure-activity data was used to build a ligand-based model of the enzyme active site, which can be used to guide the design of covalent nsP2 protease inhibitors as potential therapeutics against alphaviruses.

Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, mounting fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multi-site MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino acid peptides containing the {gamma}-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, causal agent of gray mold disease in fruits and vegetables. Its multi-site MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray-application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventative and curative suppression of gray mold disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the {gamma}-core sequence could serve as promising candidates for further development as bioinspired fungicides.

Cholesterol 24-hydroxylase (CH24H or CYP46A1) is a pivotal enzyme in brain cholesterol metabolism and has emerged as a therapeutic and imaging target in neurodegenerative disorders. Although [18F]Cholestify ([18F]CHL-2205) has shown promise as a positron emission tomography (PET) tracer for imaging of CYP46A1, the impact of cyclopropyl moiety conformation on binding and imaging performance remains unexplored. Here, we report the rational design and preliminary evaluation of novel CYP46A1 PET tracers, in which the left-side cyclopropyl group was modified into bridged, spirocyclic, and fused bicyclic architectures to probe steric and conformational effects. All compounds 9-11 exhibited high CYP46A1 affinity (IC50 = 0.19-0.28 nM). Radiosynthesis of [18F]9-11 was achieved via copper-mediated [18F]fluorination, providing practical non-decay-corrected radiochemical yields of 10-34% with excellent radiochemical purity (>98%). In vitro autoradiography in rat brain sections demonstrated specific and regionally selective binding, comparable to that observed for [18F]CHL-2205. These cyclopropyl-derived scaffolds establish a scaffold-driven strategy for PET tracer development, providing a robust framework for further structure-activity relationship studies and the rational optimization of CYP46A1 PET tracers.

Novel functionalized cyanocinnamic acid based MPC inhibitors based on pharmacologically privileged N-piperazinyl and N-piperidinyl drug templates have been synthesized for potential cancer treatment. In vitro cell proliferation inhibition studies with these derivatives 2-4 show activity in the low micromolar range. Seahorse XFe96 based mitochondrial stress tests also illustrate the ability of 2-4 to potently and acutely inhibit numerous parameters of mitochondrial respiration in MDA-MB-231, WiDr, and 4T1 cells. Further analyses of the lead compound 3 in permeabilized 4T1 cells provide evidence of specific inhibition of pyruvate driven respiration without affecting glutamate or succinate fueled respiratory processes. Combination studies with GLUT1 inhibitor BAY-876 illustrate the capacity of compound 3 to inhibit metabolic plasticity in triple negative breast cancer MDA-MB-231 cells and is synergistic in inhibiting cell proliferation in aggressive stage IV breast cancer 4T1.

Benzoxaboroles offer unusual reactivity and protein recognition for the development of small molecule drugs. Despite this potential, they are uncommon in drug discovery or in large fragment screening libraries. We synthesized a small series of structurally related benzoxaboroles containing a diazirine/alkyne tag to enable in-cell photoaffinity labeling (PAL) experiments. A subset of this library was found to have high selectivity for eukaryotic translation initiation factor 4E (eIF4E). The benzoxaborole-eIF4E interaction was found to be stereoselective in nature and competitive with the 7-methylguanosine cap of mRNA. Site of labeling experiments revealed that the benzoxaborole fragment interacts with the cap binding pocket of eIF4E. In silico modeling of the modified protein suggests that H-bonding interactions between the main chain of Trp102 and the side chain of Asn155 to the amide carbonyl and anionic boronate of the benzoxaborole, respectively, drive affinity for this challenging to drug pocket.

The increasing resistance of influenza A viruses to adamantane-based antivirals underscores the need for new inhibitors targeting both wild-type (WT) and mutant M2 ion channels. Here, we report the synthesis and biological evaluation of polycyclic cage amines designed to replace the adamantane scaffold as M2 inhibitors. These include ring-contracted and ring-expanded analogues, evaluated both as primary amines and as aryl-/heteroaryl-substituted derivatives. Most of the polycyclic amines inhibited the WT M2 channel as demonstrated by electrophysiological assays. Among them, compound 10, a 3,4,8,9-tetramethyltetracyclo[4.4.0.03..0.]decan-1-amine, emerged as a triple blocker active against M2 WT, M2 L27F, and M2 V27A channels. In contrast, compound 6c, a noradamantane-isoxazole derivative, showed selective inhibition of the S31N mutant. Although no antiviral activity was observed against influenza A virus in infected cell assays, both compounds 6c and 10 displayed significant antiviral activity against human coronavirus 229E. Furthermore, compound 10 demonstrated favourable pharmacokinetic properties. MD simulations show that noradamantane 6c binds inside the M2 S31N pore, with its ammonium forming H-bonds to Asn31 and the isoxazole positioned near Val27, restricting water entry. In contrast, larger polycyclic amines likely cannot access the pore due to steric hindrance.

We investigated a novel 4-phenoxy-quinoline-based scaffold that mislocalizes the essential mitotic kinase, AURKB. Here, we evaluated the impact of halogen substitutions (F, Cl, Br, I) on this scaffold with respect to various drug parameters. Br-substituted LXY18 was found to be a potent and orally bioavailable disruptor of cell division, at sub-nanomolar concentrations. LXY18 prevents cytokinesis by blocking AURKB relocalization in mitosis and exhibits broad-spectrum antimitotic activity in vitro. With a favorable PK profile, it shows widespread tissue distribution including the blood-brain barrier penetrance and effective accumulation in tumor tissues. More importantly, it markedly suppresses tumor growth. The novel mode of action of LXY18 may eliminate some drawbacks of direct catalytic inhibition of AURKs. Successful development of LXY18 as a clinical candidate for cancer treatment could enable a new, less toxic means of antimitotic attack that avoids drug resistance mechanisms.

Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here, we developed a 2-aminopyrimidine-based macrocyclic dual EPHA2/GAK kinase inhibitor as a chemical tool to study the role of these two kinases in viral entry and assembly. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55 as a potent dual EPHA2/GAK inhibitor. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells, mainly via its EPHA2 activity, and is therefore a promising candidate for further optimization of its activity against dengue virus.

Peptide-based biopesticides represent a promising strategy for sustainable disease control in agriculture. Synthetic antifungal peptides incorporating the {gamma}-core motif of plant defensins offer multiple modes of action (MoA) and potential as biofungicides. We investigated a synthetic variant of the olive defensin OefDef1.1 for antifungal activity, structure-function relationships, and MoA against Botrytis cinerea, the necrotrophic pathogen causing gray mold. A disulfide-bridged peptide, GMAOe1C_V1*, derived from OefDef1.1 (G32-Y53) and modified with hydrophobic amino acid substitutions, inhibited B. cinerea growth in vitro and reduced lesion formation in detached leaves. Foliar application of GMAOe1C_V1* suppressed disease symptoms in pepper plants. Mechanistically, GMAOe1C_V1* rapidly permeabilized fungal plasma membranes and accumulated in vacuoles, triggering vacuolar expansion and cell death. It also inhibited protein synthesis in vitro and in vivo, suggesting a role as a translation inhibitor. Alanine scanning mutagenesis of the non-disulfide bridged variant identified the 7RHSKH11 motif as essential for antifungal activity. Circular dichroism revealed an unstructured conformation with minimal secondary structure. Transcriptomic analysis of GMAOe1C_V1* treated B. cinerea germlings showed downregulation of genes involved in mitochondrial function and amino acid biosynthesis. These findings demonstrate the potential of an olive defensin-derived peptide as a bio-inspired antifungal agent with multifaceted MoA, supporting its development for crop protection.

Dysregulation of the Wnt1/{beta}-catenin signaling pathway has been demonstrated to be a driving factor in the propagation of several human cancers. Previous studies have discovered methyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate (MSAB) as a selective inhibitor of the Wnt1/{beta}-catenin signaling pathway, which putatively functions through direct engagement of {beta}-catenin. To understand how changes to the identity and position of the methyl ester affect the in vitro potency of this compound in Wnt1-driven mammalian cell lines, we prepared and evaluated three analogs of MSAB with 3- and 4-substituted methyl and ethyl esters. In MTT assays, analogs with methyl esters showed significantly more activity than their ethyl ester counterparts and both 4-substituted esters exhibited significantly attenuated antiproliferative activity, with MSAB exhibiting dose-dependent activity across cancerous cell lines. Further analysis by flow cytometry reveals low-Annexin V signal, suggesting that these compounds do not function via a pro-apoptotic pathway. Additionally, through a TCF/LEF-activated luciferase reporter cell assay, we observe that the 4-substituted methyl ester analogous to MSAB exhibits slightly diminished Wnt1-inhibitory activity, while 3- and 4-substituted ethyl esters exhibit minimal Wnt1-inhibitory activity. This difference in potency with a simple ester substitution might be attributed to several factors that ultimately drive antiproliferative activity, prompting the investigation of other potential substituents to further investigate the structure-activity relationship of these compounds as Wnt1-based antiproliferative agents.

The emergence of antibiotic resistance necessitates the discovery of new scaffolds with improved efficacy and drug-like properties. In this work, a new series of acid-functionalized carbazole derivatives was designed, synthesized, and comprehensively evaluated for their antibacterial potential. The incorporation of acidic functionalities into the carbazole framework enhanced physicochemical properties and biological interactions, yielding distinct strain-specific antibacterial activities. Minimum inhibitory concentration (MIC) studies demonstrated that 3-methyl-1,4-dioxo-4,9-dihydro-1H-carbazole-6-carboxylic acid (2) exhibited broad-spectrum potency, particularly against S. aureus and E. coli, while 6-methyl-9H-carbazole-3-carboxylic acid (3) showed selectivity against B. cereus and (E)-3-methyl-1-(2-tosylhydrazono)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxylic acid (1) displayed strong activity toward S. typhimurium. Molecular docking studies revealed favourable binding affinities of all derivatives toward bacterial dihydrofolate reductase (DHFR), with compound 1 showing the highest docking scores. Molecular dynamics simulations further confirmed the broad conformational adaptability of compound 1, the target-specific stability of compound 3, and protein-dependent binding behaviour of compound 2. Complementary ADMET predictions indicated that all compounds adhered to Lipinskis rules, with compound 3 displaying the most favourable pharmacokinetic profile, including high oral bioavailability and low toxicity risk. Together, these experimental and computational findings establish acid-functionalized carbazole scaffolds as promising antibacterial candidates.

The 2020 SARS-CoV-2 coronavirus pandemic highlighted the urgent need for novel small molecule antiviral drugs. (S)-x38 DNDI-6510 is a non-covalent SARS-CoV-2 main protease inhibitor developed by the open science collaboration COVID Moonshot. Here, we report on the metabolic and toxicologic optimization of the lead series previously disclosed by the COVID Moonshot Initiative, leading up to the selection of (S)-x38 DNDI-6510 as the preclinical candidate. We describe the thorough profiling of the series, identifying key risks such as formation of genotoxic metabolites and high clearance, which were successfully addressed during lead optimization. In addition, we disclose the in vitro and in vivo evaluation of (S)-x38 DNDI-6510 in pharmacokinetic and pharmacodynamic models, exploring multiple approaches to ameliorate rodent-specific metabolic clearance, and show that both co-dosing of (S)-x38 DNDI-6510 with an ABT inhibitor and utilizing a metabolically humanized mouse model (8HUM) achieve significant improvements in exposure. Through comparisons of ABT co-dosing and humanized mouse models in efficacy experiments, we demonstrate that continuous exposure over cellular EC90 is required for SARS-CoV-2 antiviral efficacy in vivo in an antiviral model using a mouse-adapted SARS-CoV-2 strain. Finally, (S)-x38 DNDI-6510 was assessed in maximum tolerated dose experiments in two species, demonstrating significant in vivo PXR-linked auto-induction of metabolism, leading to the discontinuation of this compound. In summary, we report the successful effort to overcome series-specific AMES liabilities in a lead development program. Downstream optimization of existing series will require in-depth optimization of rodent-specific liabilities and metabolic induction profile.

STK10 (serine/threonine kinase 10, LOK), is an important regulator of diverse cellular processes, such as cell cycle progression or lymphocyte migration. STK10 has emerged as a potential therapeutic target for diseases associated with impaired cell migration and cell division. Here we present a late-stage optimization of a macrocyclic pyrazolo[1,5-a]pyrimidine scaffold that led to a urea-based lead series targeting the back-pocket of STK10. Co-crystal structure analysis of 23 revealed that the optimized macrocycles adopted a unique binding mode that protrudes deep into the back pocket of STK10. Compound 23 exhibited potent on-target activity in biophysical and activity assays and displayed nanomolar activity for STK10 in cells. In addition, 23 shows good selectivity against the kinome and remarkably also against the closely related kinase SLK (STE20-like kinase). Therefore, we propose that targeting the unique and largely extended pocket in STK10 represents an opportunity to develop highly selective STK10 inhibitors. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/666149v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@36f039org.highwire.dtl.DTLVardef@d55025org.highwire.dtl.DTLVardef@80c007org.highwire.dtl.DTLVardef@bf2471_HPS_FORMAT_FIGEXP M_FIG C_FIG

Blockade of the TGF{beta} signalling pathway has emerged from preclinical studies as a potential treatment to enhance the efficacy of immune checkpoint inhibition in advanced colorectal cancer (CRC) and several other types of cancer. However, clinical translation of first-generation inhibitors has known little success. Here, we report the synthesis and characterization of HYL001, a potent inhibitor of TGF{beta} receptor 1 (ALK5), that is approximately 9 times more efficacious than the structurally related compound galunisertib, while maintaining a favourable safety profile. HYL001 in combination with immune checkpoint blockade (anti-PD1) eradicates liver metastases generated in mice by microsatellite stable, aggressive colorectal cancer tumours at doses where galunisertib is ineffective. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/593510v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1909963org.highwire.dtl.DTLVardef@4644d2org.highwire.dtl.DTLVardef@1506d80org.highwire.dtl.DTLVardef@14504b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Reprogrammed metabolism of cancer cells offers a unique target for pharmacological intervention. In the current study, a series of novel and potentially metabolically stable fluoro-substituted aminocarboxycoumarin derivatives are evaluated for their mitochondrial pyruvate carrier (MPC) inhibition properties. Our studies indicate that the aminocarboxycoumarin template elicits potent MPC inhibitory characteristics, and specifically, structure activity relationship studies show that the N-methyl-N-benzyl structural template provides the optimal inhibitory capacity. Further respiratory experiments demonstrate that candidate compounds specifically inhibit pyruvate driven respiration without substantially affecting other metabolic fuels consistent with MPC inhibition. Further, computational homology and inhibitor docking studies illustrate that aminocarboxycoumarin binding characteristics are indicative of reversible covalent bonding with amino acids in the pyruvate binding domain. Epifluorescent microscopy experiments illustrated that FACC2 accumulates in the mitochondria to a similar extent as parent 7ACC2. Additionally, lead candidate aminocarboxycoumarin derivative D7 elicits cancer cell proliferation inhibition specifically in monocarboxylate transporter 1 (MCT1) expressing 4T1, consistent with its ability to accumulate intracellular lactate. In vivo tumor growth studies illustrate that D7 significantly reduces the tumor burden in two isogeneic murine cell lines 4T1 and 67nr. These studies provide novel MPC inhibitors with potential for anticancer applications.

Focal Adhesion Kinase (FAK) is a non-receptor protein tyrosine kinase that plays a crucial role in various oncogenic processes related to cell adhesion, migration, proliferation, and survival. The strategic targeting of FAK represents a burgeoning approach to address resistant tumors, such as pancreatic ductal adenocarcinoma (PDAC). Herein, we report a new series of twenty imidazo[2,1-b][1,3,4]thiadiazole derivatives assayed for their antiproliferative activity against the National Cancer Institute (NCI-60) panel and a wide panel of PDAC models. Lead compound 10l exhibited effective antiproliferative activity against immortalized (SUIT-2, CAPAN-1, PANC-1, PATU-T, BxPC-3), primary (PDAC-3) and gemcitabine-resistant clone (PANC-1-GR) PDAC cells, eliciting IC50 values in the low micromolar range (1.04-3.44 {micro}M), associated with a significant reduction in cell-migration and spheroid shrinkage in vitro. High-throughput kinase arrays revealed a significant inhibition of the FAK signalling network, associated to induction of cell cycle arrest in G2/M phase, suppression of tumor cell invasion and apoptosis induction. The low selectivity index/toxicity prompted studies using PDAC mouse xenografts, demonstrating significant inhibition of tumor growth and safety. In conclusion, compound 10l displayed antitumor activity and safety in both in vitro and in vivo models, emerging as a highly promising lead for the development of FAK inhibitors in PDAC.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/582899v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@89ffd0org.highwire.dtl.DTLVardef@1f9cba2org.highwire.dtl.DTLVardef@a821b3org.highwire.dtl.DTLVardef@3c7b6a_HPS_FORMAT_FIGEXP M_FIG C_FIG Triple-negative breast cancer (TNBC) represents the most aggressive form among breast carcinoma subtypes. Due to limited therapy options, identification of novel active pharmacological compounds is an urgent medical need. A promising approach in cancer treatment is the pharmacological inhibition of murine double minutes 2 (MDM2)-p53/p73 interactions inducing apoptosis in tumors. We here describe a novel bipyrimidineamide based -helix mimetic 9 (VWK603) which was designed as a lead candidate to target MDM2. 9 (VWK603) potently induced cell death in the TNBC cell lines MDA-MB-231, MDA-MB-436 and MDA-MB-468 with IC50 values ranging between 3.7 {micro}M and 6.6 {micro}M. The anti-tumor activity was about four more potent higher than determined for the MDM2-specific inhibitor Nutlin-3a. Mechanistic analysis revealed induction of cellular apoptosis as the underlying mode of action of 9 (VWK603) anti-tumor activity. Since toxicity was observed to be reduced in non-cancerous breast cells, these studies make 9 (VWK603) a promising candidate for further preclinical MDM2 inhibitor development.

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception.1 However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs.1-2 In this paper, we describe the development and mechanism of action of new type III inhibitors that bind CDK2 with nanomolar affinity, making them the highest affinity, structurally confirmed allosteric CDK inhibitors reported. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as efficacious contraceptive agents is seen by incubation with mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.

KRAS mutation occurs in nearly 30% of human cancers, yet the most prevalent and oncogenic KRAS mutation (G12D) still lacks inhibitors. Herein, we explored the formation of a salt-bridge between KRASs Asp12 residue and a series of potent inhibitors. Our ITC results show that these inhibitors bind to and inhibit both GDP-bound and GTP-bound KRAS G12D, and our crystallographic studies revealed the structural basis of inhibitor binding in the switch-II pocket, experimentally confirming the formation of a salt-bridge between the piperazine moiety of the inhibitors and the 12D residue of the mutant protein. Among KRAS family proteins and mutants, both ITC and enzymatic assays support the selectivity of the inhibitors for KRAS G12D, and the inhibitors disrupt the KRAS-CRAF interaction. We also observed inhibition of cancer cell proliferation and inhibition of MAPK signaling by a representative inhibitor (TH-Z835); however, since this was not fully dependent on KRAS mutation status, it is possible that our inhibitors may have off-target effects via non-KRAS small GTPases. Experiments with a mouse model of pancreatic cancer showed that TH-Z835 significantly reduced tumor volume and synergized with an anti-PD-1 antibody. Collectively, our study demonstrates proof-of-concept for a salt-bridge, induced-fit pocket strategy for KRAS G12D, which warrants future medicinal chemistry efforts for optimal efficacy and minimized off-target effects.