Small Molecule Compounds Research Articles

Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

BackgroundGlioblastoma (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some GBMs, cell proliferation is fueled by a transcriptional regulator, repressor element-1 silencing transcription factor (REST).ResultsUsing CRISPR/Cas9, we identified GBM cell lines dependent on REST activity. We developed new small molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) to reduce REST protein level and transcriptional activity in glioblastoma cells. Top leads of the series like GR-28 exhibit potent cytotoxicity, reduce REST protein level, and suppress its transcriptional activity. Upon the loss of REST protein, GBM cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation using long-chain acyl-CoA synthetase inhibitor Triacsin C demonstrated substantial synergetic potential without inducing hepatotoxicity.ConclusionsOur results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST GBM.

The insulin-like growth factor binding protein-microfibrillar associated protein-sterol regulatory element binding protein axis regulates fibroblast-myofibroblast transition and cardiac fibrosis.

Excessive fibrogenesis is associated with adverse cardiac remodelling and heart failure. The myofibroblast, primarily derived from resident fibroblast, is the effector cell type in cardiac fibrosis. Megakaryocytic leukaemia 1 (MKL1) is considered the master regulator of fibroblast-myofibroblast transition (FMyT). The underlying transcriptional mechanism is not completely understood. Our goal was to identify novel transcriptional targets of MKL1 that might regulate FMyT and contribute to cardiac fibrosis. RNA sequencing (RNA-seq) performed in primary cardiac fibroblasts identified insulin-like growth factor binding protein 5 (IGFBP5) as one of the genes most significantly up-regulated by constitutively active (CA) MKL1 over-expression. IGFBP5 expression was detected in heart failure tissues using RT-qPCR and western blots. Once activated, IGFBP5 translocated to the nucleus to elicit a pro-FMyT transcriptional programme. Consistently, IGFBP5 knockdown blocked FMyT in vitro and dampened cardiac fibrosis in mice. Of interest, IGFBP5 interacted with nuclear factor of activated T-cell 4 (NFAT4) to stimulate the transcription of microfibril-associated protein 5 (MFAP5). MFAP5 contributed to FMyT and cardiac fibrosis by enabling sterol response element binding protein 2 (SREBP2)-dependent cholesterol synthesis. Our data unveil a previously unrecognized transcriptional cascade, initiated by IGFBP5, that promotes FMyT and cardiac fibrosis. Screening for small-molecule compounds that target this axis could yield potential therapeutics against adverse cardiac remodelling.

Mechanisms of Sepsis-Induced Acute Lung Injury and Advancements of Natural Small Molecules in Its Treatment

Sepsis-induced acute lung injury (ALI), characterized by widespread lung dysfunction, is associated with significant morbidity and mortality due to the lack of effective pharmacological treatments available clinically. Small-molecule compounds derived from natural products represent an innovative source and have demonstrated therapeutic potential against sepsis-induced ALI. These natural small molecules may provide a promising alternative treatment option for sepsis-induced ALI. This review aims to summarize the pathogenesis of sepsis and potential therapeutic targets. It assembles critical updates (from 2014 to 2024) on natural small molecules with therapeutic potential against sepsis-induced ALI, detailing their sources, structures, effects, and mechanisms of action.

Abstract CT105: Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug

Abstract The human genome contains G-rich regions that are over-represented in many cancer gene promoters. These can form higher-order G-quadruplex arrangements under the influence of an appropriate and selective small molecule compound. The resulting ligand-G-quadruplex complex may be resistant to polymerase read-through and thus results in transcriptional down-regulation of target genes. QN-302, based on a tetra-substituted naphthalene diimide chemotype, is a potent and selective G-quadruplex-binding compound that has been designed and initially developed at University College London. It displays single-digit cell growth inhibition in a panel of pancreatic ductal adenocarcinoma (PDAC) cell lines. RNA-seq analysis shows that it is a pan-quadruplex agent, down-regulating the expression of multiple G-quadruplex-promoter cancer genes and pathways in PDAC. It also shows in vivo anti-tumor activity in several animal models for PDAC, with low toxicity observed at therapeutic doses. QN-302 was licensed to Qualigen Therapeutics Inc in January 2022. It was granted Orphan Drug designation by the FDA in December 2022 for PDAC. This was followed by extensive regulatory toxicological and pharmacological studies together with successful scale-up synthesis, purification and formulation. QN-302 was granted IND clearance in August 2023 by the FDA for phase 1 clinical trials in advanced or metastatic solid tumors. This Phase 1a clinical trial is being conducted in two USA cancer centers (START Midwest, Grand Rapids, MI and Honor Health, Scottsdale, AZ) using a protocol based on 28-day once-weekly dose escalation and evaluation of three potential biomarkers, two of which have been suggested by transcriptomic analysis as being indicative of G-quadruplex-induced drug response and accompanying decrease of anti-tumor activity. So far 1 Cohort (3 patients) has been dosed at the lowest dose of 1.62 mg of QN-302 as part of this Phase 1a clinical trial. Patient #1, a male patient of metastatic pancreatic cancer, is currently in Cycle 4 of therapy showing Stable Disease and no AEs or SAEs reported. Patient #2, a female patient of metastatic colorectal cancer, completed Cycle 2 after which she showed progression upon CT Scan before entering Cycle 3 and was taken off study. Patient #3, a male patient of metastatic PDAC, is currently ending Cycle 2, about to enter Cycle 3, showing Stable Disease upon CT scan. All patients dosed showed no AEs, SAEs or side effects. They were also clinically observed to have improved QOL compared to prior SOC while on therapy with QN-302, This presentation will outline this unique approach and summarize safety, tolerability, PK/PD data and any early clinical data as described above including preliminary biomarker data now that clinical evaluation has commenced. The advantages and challenges of QN-302 will also be discussed. Citation Format: Tariq Arshad, Stephen Neidle, Sreenivasa Chandana, Erkut Borazanci. Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT105.

Prognostic Model Construction of Disulfidptosis-Related Genes and Targeted Anticancer Drug Research in Pancreatic Cancer.

Pancreatic cancer stands as one of the most lethal malignancies, characterized by delayed diagnosis, high mortality rates, limited treatment efficacy, and poor prognosis. Disulfidptosis, a recently unveiled modality of cell demise induced by disulfide stress, has emerged as a critical player intricately associated with the onset and progression of various cancer types. It has emerged as a promising candidate biomarker for cancer diagnosis, prognosis assessment, and treatment strategies. In this study, we have effectively established a prognostic risk model for pancreatic cancer by incorporating multiple differentially expressed long non-coding RNAs (DElncRNAs) closely linked to disulfide-driven cell death. Our investigation delved into the nuanced relationship between the DElncRNA-based predictive model for disulfide-driven cell death and the therapeutic responses to anticancer agents. Our findings illuminate that the high-risk subgroup exhibits heightened susceptibility to the small molecule compound AZD1208, positioning it as a prospective therapeutic agent for pancreatic cancer. Finally, we have elucidated the underlying mechanistic potential of AZD1208 in ameliorating pancreatic cancer through its targeted inhibition of the peroxisome proliferator-activated receptor-γ (PPARG) protein, employing an array of comprehensive analytical methods, including molecular docking and molecular dynamics (MD) simulations. This study explores disulfidptosis-related genes, paving the way for the development of targeted therapies for pancreatic cancer and emphasizing their significance in the field of oncology. Furthermore, through computational biology approaches, the drug AZD1208 was identified as a potential treatment targeting the PPARG protein for pancreatic cancer. This discovery opens new avenues for exploring targets and screening drugs for pancreatic cancer.

Potential shared pathogenic mechanisms between endometriosis and inflammatory bowel disease indicate a strong initial effect of immune factors

IntroductionOver the past decades, immune dysregulation has been consistently demonstrated being common charactoristics of endometriosis (EM) and Inflammatory Bowel Disease (IBD) in numerous studies. However, the underlying pathological mechanisms remain unknown. In this study, bioinformatics techniques were used to screen large-scale gene expression data for plausible correlations at the molecular level in order to identify common pathogenic pathways between EM and IBD.MethodsBased on the EM transcriptomic datasets GSE7305 and GSE23339, as well as the IBD transcriptomic datasets GSE87466 and GSE126124, differential gene analysis was performed using the limma package in the R environment. Co-expressed differentially expressed genes were identified, and a protein-protein interaction (PPI) network for the differentially expressed genes was constructed using the 11.5 version of the STRING database. The MCODE tool in Cytoscape facilitated filtering out protein interaction subnetworks. Key genes in the PPI network were identified through two topological analysis algorithms (MCC and Degree) from the CytoHubba plugin. Upset was used for visualization of these key genes. The diagnostic value of gene expression levels for these key genes was assessed using the Receiver Operating Characteristic (ROC) curve and Area Under the Curve (AUC) The CIBERSORT algorithm determined the infiltration status of 22 immune cell subtypes, exploring differences between EM and IBD patients in both control and disease groups. Finally, different gene expression trends shared by EM and IBD were input into CMap to identify small molecule compounds with potential therapeutic effects.Results113 differentially expressed genes (DEGs) that were co-expressed in EM and IBD have been identified, comprising 28 down-regulated genes and 86 up-regulated genes. The co-expression differential gene of EM and IBD in the functional enrichment analyses focused on immune response activation, circulating immunoglobulin-mediated humoral immune response and humoral immune response. Five hub genes (SERPING1、VCAM1、CLU、C3、CD55) were identified through the Protein-protein Interaction network and MCODE.High Area Under the Curve (AUC) values of Receiver Operating Characteristic (ROC) curves for 5hub genes indicate the predictive ability for disease occurrence.These hub genes could be used as potential biomarkers for the development of EM and IBD. Furthermore, the CMap database identified a total of 9 small molecule compounds (TTNPB、CAY-10577、PD-0325901 etc.) targeting therapeutic genes for EM and IBD.DiscussionOur research revealed common pathogenic mechanisms between EM and IBD, particularly emphasizing immune regulation and cell signalling, indicating the significance of immune factors in the occurence and progression of both diseases. By elucidating shared mechanisms, our study provides novel avenues for the prevention and treatment of EM and IBD.

Efficient Delivery of Lomitapide using Hybrid Membrane-Coated Tetrahedral DNA Nanostructures for Glioblastoma Therapy.

Glioblastoma (GBM) is the most aggressive and prevalent primary malignant tumor of the central nervous system. Traditional chemotherapy has poor therapeutic effects and significant side effects due to drug resistance, the natural blood-brain barrier (BBB), and nonspecific distribution, leading to a lack of clinically effective therapeutic drugs. Here, 1430 small molecule compounds are screened based on a high-throughput drug screening platform and a novel anti-GBM drug, lomitapide (LMP) is obtained. Furthermore, a bionic nanodrug delivery system (RFA NPs) actively targeting GBM is constructed, which mainly consists of tetrahedral DNA nanocages (tFNA NPs) loaded with LMP as the core and a folate-modified erythrocyte-cancer cell-macrophage hybrid membrane (FRUR) as the shell. FRUR camouflage conferred unique features on tFNA NPs, including excellent biocompatibility, improved pharmacokinetic profile, efficient BBB permeability, and tumor targeting ability. The results show that the LMP RFA NPs exhibited superior and specific anti-GBM activities, reduced off-target drug delivery, prolonged lifespan, and has negligible side effects in tumor-bearing mice. This study combines high-throughput drug screening with biomimetic nanodrug delivery system technology to provide a theoretical and practical basis for drug development and the optimization of clinical treatment strategies for GBM treatment.

KRAS: Biology, Inhibition, and Mechanisms of Inhibitor Resistance

KRAS is a small GTPase that is among the most commonly mutated oncogenes in cancer. Here, we discuss KRAS biology, therapeutic avenues to target it, and mechanisms of resistance that tumors employ in response to KRAS inhibition. Several strategies are under investigation for inhibiting oncogenic KRAS, including small molecule compounds targeting specific KRAS mutations, pan-KRAS inhibitors, PROTACs, siRNAs, PNAs, and mutant KRAS-specific immunostimulatory strategies. A central challenge to therapeutic effectiveness is the frequent development of resistance to these treatments. Direct resistance mechanisms can involve KRAS mutations that reduce drug efficacy or copy number alterations that increase the expression of mutant KRAS. Indirect resistance mechanisms arise from mutations that can rescue mutant KRAS-dependent cells either by reactivating the same signaling or via alternative pathways. Further, non-mutational forms of resistance can take the form of epigenetic marks, transcriptional reprogramming, or alterations within the tumor microenvironment. As the possible strategies to inhibit KRAS expand, understanding the nuances of resistance mechanisms is paramount to the development of both enhanced therapeutics and innovative drug combinations.

Mechanism Research of PZD Inhibiting Lung Cancer Cell Proliferation, Invasion, and Migration Based on Network Pharmacology.

A classic Chinese medicine decoction, Pinellia ternata (Thunb.) Breit-Zingiber officinale Roscoe (Ban-Xia and Sheng-Jiang in Chinese) decoction (PZD), has shown significant therapeutic effects on lung cancer. This study aimed to explore and elucidate the mechanism of action of PZD on lung cancer using network pharmacology methods. Active compounds were selected according to the ADME parameters recorded in the TCMSP database. Potential pathways related to genes were identified through GO and KEGG analysis. The compound target network was constructed by using Cytoscape 3.7.1 software, and the core common targets were obtained by protein-protein interaction (PPI) network analysis. Batch molecular docking of small molecule compounds and target proteins was carried out by using the AutoDock Vina program. Different concentrations of PZD water extracts (10, 20, 40, 80, and 160 μg/mL) were used on lung cancer cells. Moreover, MTT and Transwell experiments were conducted to validate the prominent therapeutic effects of PZD on lung cancer cell H1299. A total of 381 components in PZD were screened, of which 16 were selected as bioactive compounds. The compound-target network consisting of 16 compounds and 79 common core targets was constructed. MTT experiment showed that the PZD extract could inhibit the cell proliferation of NCI-H1299 cells, and the IC50 was calculated as 97.34 ± 6.14 μg/mL. Transwell and wound-healing experiments showed that the PZD could significantly decrease cell migration and invasion at concentrations of 80 and 160 μg/mL, respectively. The in vitro experiments confirmed that PZD had significant therapeutic effects on lung cancer cells, mainly through the PI3K/AKT signaling pathway. PZD could inhibit the cell proliferation, migration, and invasion of NCI-H1299 cells partially through the PI3K/AKT signaling pathway. These findings suggested that PZD might be a potential treatment strategy for lung cancer patients.

Effect of small-molecule phenolic compounds on the removal of triclosan (TCS) by heat/persulfate process: Formation of cross-coupling products and implications for wastewater treatment

In this study, the effect of three small molecule phenolic compounds (SMPCs) (Phenol (PHOH), p-hydroxybenzoic acid (PHBA) and 3,5-dihydroxytoluene (DHT)) on the transformation of triclosan (TCS) in heat activated persulfate (heat/PS) process was investigated. TCS in single and mixed solutions were almost completely removed after 120 min of heat/PS treatment. Compared with single TCS system, the generation of cross-coupling products in the mixed system was beneficial for the removal of total organic carbon (TOC). Overall cytotoxicity and genotoxicity tests on mammalian cells showed that the reaction products in mixed phenol system was less toxic than those in the single TCS system, which confirmed the low toxicity of cross-coupling products. Product analysis was combined with theoretical calculations to propose a mechanism for the cross-coupling of coexisting phenolic pollutants that hydrogen on the phenolic hydroxyl groups of different pollutants was extracted by SO4•- to form phenoxy radicals, which coupled with each other to form cross-coupling products. Lowering the pH of the reaction solution can promote the occurrence of cross-coupling reaction, favoring the removal of TOC and the reduction of toxicity. These results will provide insights into the transformation of phenolic pollutants in water treatment for further control of environmental risks.

Increased Vulnerability to Ferroptosis in FUS-ALS

Ferroptosis, a regulated form of cell death characterized by iron-dependent lipid peroxide accumulation, plays a pivotal role in various pathological conditions, including neurodegenerative diseases. While reasonable evidence for ferroptosis exists, e.g., in Parkinson’s disease or Alzheimer’s disease, there are only a few reports on amyotrophic lateral sclerosis (ALS), a fast progressive and incurable neurodegenerative disease characterized by progressive motor neuron degeneration. Interestingly, initial studies have suggested that ferroptosis might be significantly involved in ALS. Key features of ferroptosis include oxidative stress, glutathione depletion, and alterations in mitochondrial morphology and function, mediated by proteins such as GPX4, xCT, ACSL4 FSP1, Nrf2, and TfR1. Induction of ferroptosis involves small molecule compounds like erastin and RSL3, which disrupt system Xc− and GPX4 activity, respectively, resulting in lipid peroxidation and cellular demise. Mutations in fused in sarcoma (FUS) are associated with familial ALS. Pathophysiological hallmarks of FUS-ALS involve mitochondrial dysfunction and oxidative damage, implicating ferroptosis as a putative cell-death pathway in motor neuron demise. However, a mechanistic understanding of ferroptosis in ALS, particularly FUS-ALS, remains limited. Here, we investigated the vulnerability to ferroptosis in FUS-ALS cell models, revealing mitochondrial disturbances and increased susceptibility to ferroptosis in cells harboring ALS-causing FUS mutations. This was accompanied by an altered expression of ferroptosis-associated proteins, particularly by a reduction in xCT expression, leading to cellular imbalance in the redox system and increased lipid peroxidation. Iron chelation with deferoxamine, as well as inhibition of the mitochondrial calcium uniporter (MCU), significantly alleviated ferroptotic cell death and lipid peroxidation. These findings suggest a link between ferroptosis and FUS-ALS, offering potential new therapeutic targets.

Guanidine-to-piperidine switch affords high affinity small molecule NPFF ligands with preference for NPFF1-R and NPFF2-R subtypes

The Neuropeptide FF (NPFF) receptor system is known to modulate opioid actions and has been shown to mediate opioid-induced hyperalgesia and tolerance. The lack of subtype selective small molecule compounds has hampered further exploration of the pharmacology of this receptor system. The vast majority of available NPFF ligands possess a highly basic guanidine group, including our lead small molecule, MES304. Despite providing strong receptor binding, the guanidine group presents a potential pharmacokinetic liability for in vivo pharmacological tool development. Through structure-activity relationship exploration, we were able to modify our lead molecule MES304 to arrive at guanidine-free NPFF ligands. The novel piperidine analogues 8b and 16a are among the few non-guanidine based NPFF ligands known in literature. Both compounds displayed nanomolar NPFF-R binding affinity approaching that of the parent molecule. Moreover, while MES304 was non-subtype selective, these two analogues presented new starting points for subtype selective scaffolds, whereby 8b displayed a 15-fold preference for NPFF1-R, and 16a demonstrated an 8-fold preference for NPFF2-R. Both analogues showed no agonist activity on either receptor subtype in the in vitro functional activity assay, while 8b displayed antagonistic properties at NPFF1-R. The calculated physicochemical properties of 8b and 16a were also shown to be more favorable for in vivo tool design. These results indicate the possibility of developing potent, subtype selective NPFF ligands devoid of a guanidine functionality.

Abstract 6576: Discovery of potent and selective novel small molecule drugs to target estrogen receptor mutants in endocrine-resistant breast cancer

Abstract Introduction: The acquisition of estrogen receptor alpha (ERα) gene (ESR1) mutations is a key driver for the development of resistance to current endocrine therapy in breast cancer. Clinical studies have shown that ESR1 mutations are frequently observed in metastatic ER-positive breast cancer patients and are associated with poor survival. It is well appreciated that activating ESR1 somatic mutations, especially Y537S and D538G, can drive estrogen-independent activities. In addition, these ESR1 mutations diminish the potency of the current standard-of-care agents (tamoxifen and fulvestrant) that bind ERα directly. Therefore, it is an unmet need to develop next-generation antiestrogens that inhibit ERα mutant signaling in breast cancer to improve patient survival. Here, we search for small molecule inhibitors against ERα mutants Y537S and D538G using DNA-encoded chemical library (DEL). Methods: ERα ligand binding domain (LBD) proteins corresponding to wild type (WT), Y537S and D538G mutants were expressed in E. coli and purified by Ni-NTA, anion exchange, and size exclusion chromatography. The ability of these purified proteins to bind ligands was tested in biochemical assays to validate their use in DEL selections. We conducted a DEL affinity selection using our in-house collection of 6 billion small molecule compounds against WT, Y537S and D538G mutants ER LBD proteins in the presence and absence of estradiol. Selection hits that enriched with these targets were resynthesized off-DNA and tested in biochemical and functional studies using CRISPR-Cas9 knock-in Y537S or D538G mutant breast cancer cells. Results: We have successfully purified microgram amounts of ERα LBD for WT, Y537S, and D538G proteins. In our biochemical assay, the SRC3 peptide binds to the WT ERα LBD in the presence of estradiol, whereas Y537S and D538G LBDs bind the SRC3 peptide in the absence of estradiol, consistent with these mutants constitutively binding to SRC3. Our multibillion small molecule collections of DELs can screen and identify several hits in WT and mutant ERα LBDs. To confirm the selection output, we synthesized off-DNA compounds and validated these in biochemical and cell-based studies. Compounds CDD-1274 and CDD-1802 dramatically decrease the WT and mutant ERα protein levels in multiple ER+ breast cancer cells. These compounds greatly reduce protein levels for GREB1, TFF1, c-MYC, E2F1, and survivin in wildtype and mutant breast cancer cells, and they increase levels of cleaved PARP, a marker for apoptosis in wildtype and mutant ER+ breast cancer cells but not in ER-negative breast cancer cells. Conclusion: Our study rapidly identified potent and novel small molecule drugs to target estrogen receptor mutants using our DNA-encoded chemical library platform. Acknowledgments: Funding sources (NIH/NCI R03 CA259664 and CPRIT RP220524 to MP). Citation Format: Anil Kumar Devakrishnan, Chandrashekhar Madasu, Suzanne A.W Fuqua, Martin M. Matzuk, Murugesan Palaniappan. Discovery of potent and selective novel small molecule drugs to target estrogen receptor mutants in endocrine-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6576.

Abstract 5776: Target identification, selectivity profiling and binding site mapping of small molecule and peptide drugs by LiP-MS

Abstract Target identification in the cellular context is a critical step for both target-based and phenotypic drug discovery. Limited proteolysis coupled with mass spectrometry (LiP-MS) has emerged as a powerful technique for target deconvolution of small molecules or peptides in cell lysate without compound modification or genetic manipulation of cell lines [1,2,3,4]. Utilizing a non-specific protease under well-controlled conditions, LiP-MS exploits drug-induced structural alteration or steric hindrance on protein targets and utilizes quantitative mass spectrometry to probe over 250’000 peptides covering more than 9000 proteins in the proteome. To aid target ID, this workflow incorporates a 7-concentration dose response experiment and a machine-learning framework [1] to compute a LiP-score to rank identified target proteins, as well as predict potential binding site. In this study, we present target deconvolution results on four small molecule compounds and one peptide compound with very distinct pharmacological profile: 1) staurosporine, a broad-specific kinase inhibitor; 2) roniciclib, a failed clinical-stage pan-CDK inhibitor; 3) selumetinib, an allosteric, non-ATP-competitive MEK 1 and 2 inhibitor; 4) NST-628, a newly developed non-degradating molecular glue inhibiting the RAS/MAPK pathway; and 5) Acetyl-calpastatin, a 24 amino-acid polypeptide inhibitor of calpain 1 and 2. Target ID experiment with staurosporine and roniciclib yielded > 150 and 68 kinase targets were identified respectively. For the pan-kinase inhibitor roniciclib, both cell-cycle CDKs (CDK1/cyclin B, CDK2/cyclin E, CDK4 cyclin D) and transcriptional CDK9 were among the targets. For Selumetinib, LiP-score ranking identified MEK1 and MEK2 as the top two targets, and only kinases among a total of 23 targets. Furthermore, mapping the LiP-peptides from this experiment onto MEK1 showed that they are in close proximity to the selumetinib allosteric binding site on co-crystal structure. Last but not least, we assessed the target landscape of NST-628, a novel non-degrading molecular glue and identified MEK1 and MEK2 clearly as the primary targets. This finding is in line with orthogonal data obtained from X-ray crystallography [5]. Collectively, this data demonstrates that LiP-MS can be deployed to effectively identify protein drug targets and predict binding sites in complex cellular millieu independent of the compound’s mechanism of action and without compound modification or labeling. These capabilities make LiP-MS a powerful addition to the target deconvolution toolbox. [1] Piazza and Beaton et al. Nature Comm 2020 [2] Hendricks and Beaton et al. ACS Chem Bio 2021 [3] AACR 2022 [4] AACR 2023 [5] AACR-NCI-EORTC 2023 Citation Format: Fabio Sabino, Martin Soste, Dominique Kamber, Maria Stella Ritorto, Klaus Hoeflich, Michael Hale, Bradley Quade, Xin Huang, Nigel Beaton, Roland Bruderer, Yuehan Feng, Lukas Reiter. Target identification, selectivity profiling and binding site mapping of small molecule and peptide drugs by LiP-MS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5776.

Mutagenesis and functional analysis of the varicella-zoster virus portal protein.

Herpesviruses replicate by cleaving concatemeric dsDNA into single genomic units that are packaged through an oligomeric portal present in preformed procapsids. In contrast to what is known about phage portal proteins, details concerning herpesvirus portal structure and function are not as well understood. A panel of 65 Varicella-Zoster virus (VZV) recombinant portal proteins with five amino acid in-frame insertions were generated by random transposon mutagenesis of the VZV portal gene, ORF54. Subsequently, 65 VZVLUC recombinant viruses (TNs) were generated via recombineering. Insertions were mapped to predicted portal domains (clip, wing, stem, wall, crown, and β-hairpin tunnel-loop) and recombinant viruses were characterized for plaque morphology, replication kinetics, pORF54 expression, and classified based on replication in non-complementing (ARPE19) or complementing (ARPE54C50) cell lines. The N- and C-termini were tolerant to insertion mutagenesis, as were certain clip sub-domains. The majority of mutants mapping to the wing, wall, β-hairpin tunnel loop, and stem domains were lethal. Elimination of the predicted ORF54 start codon revealed that the first 40 amino acids of the N-terminus were not required for viral replication. Stop codon insertions in the C-terminus showed that the last 100 amino acids were not required for viral replication. Lastly, a putative protease cleavage site was identified in the C-terminus of pORF54. Cleavage was likely orchestrated by a viral protease; however, processing was not required for DNA encapsidation and viral replication. The panel of recombinants should prove valuable in future studies to dissect mammalian portal structure and function.IMPORTANCEThough nucleoside analogs and a live-attenuated vaccine are currently available to treat some human herpesvirus family members, alternate methods of combating herpesvirus infection could include blocking viral replication at the DNA encapsidation stage. The approval of Letermovir provided proof of concept regarding the use of encapsidation inhibitors to treat herpesvirus infections in the clinic. We propose that small-molecule compounds could be employed to interrupt portal oligomerization, assembly into the capsid vertex, or affect portal function/dynamics. Targeting portal at any of these steps would result in disruption of viral DNA packaging and a decrease or absence of mature infectious herpesvirus particles. The oligomeric portals of herpesviruses are structurally conserved, and therefore, it may be possible to find a single compound capable of targeting portals from one or more of the herpesvirus subfamilies. Drug candidates from such a series would be effective against viruses resistant to the currently available antivirals.

Abstract 5889: An immunoproteasome activator that expands the MHC-class I immunopeptidome, unmasks neoantigens and enhances antimyeloma T-cell activity

Abstract A hallmark of cancer is the ability to evade immunosurveillance mechanisms and thwart the efficacy of immunotherapeutic agents. The proteasome functions as an essential component of immunosurveillance by generating peptides from intracellular proteins that are then presented to T-cells. Antigens generated by the proteasome promote the infiltration of immune cells into tumors and improve tumor regression in response to immunotherapy. Cancer cells can evade elimination by the immune system by deregulating the antigen presentation machinery to downregulate the expression of proteins recognized by immune cells as antigens, creating an immunosuppressive microenvironment. In contrast to constitutive proteasomes, immunoproteasomes are a highly specialized proteasome variant that are highly expressed in antigen-presenting cells. Immunoproteasomes contain distinct catalytic subunits with active site substrate specificities distinct from constitutive proteasomes. Immunoproteasome degrade intracellular proteins to generate antigenic peptides that are the presented by MHC-class I (MHC-I) molecules on tumor cells for recognition by cytotoxic T-cells. We hypothesized that immunoproteasome activation could concomitantly increase the relative abundance and diversity of MHC-I antigens presented on multiple myeloma (MM) cells. High-throughput screening identified a novel small molecule (Compound A) that selectively increased immunoproteasome catalytic activity. Global proteomic integral stability assays determined that Compound A binds the proteasome structural subunit PSMA1 and that treatment with Compound A increased association of the proteasome activator PA28α/β (PSME1/2) activator with immunoproteasomes. The effect of Compound A on immunoproteasomes was abolished after the ablation of PSMA1, PSME1, or PSME2 or by the treatment of cells with suicide inhibitors that target immunoproteasome-specific catalytic sites. Treatment of MM cells with Compound A doubled the total number of MHC-I-bound peptides and increased the level of specific MHC-bound peptides up to 200-fold. Interestingly, a number of tumor-associated antigens and neoantigens were dramatically unmasked following treatment of MM cells with the immunoproteasome activator. Importantly, the treatment of patient CD138+ cells with Compound A promoted the antimyeloma activity of allogenic and autologous patient-derived CD8+ T-cells. Taken together, our results demonstrate the paradigm-shifting translational impact of immunoproteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets to personalize T-cell-directed immunotherapy. Citation Format: Priyanka S. Rana, James J. Ignatz-Hoover, James J. Driscoll. An immunoproteasome activator that expands the MHC-class I immunopeptidome, unmasks neoantigens and enhances antimyeloma T-cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5889.

Abstract 3542: The Chemical Probes Portal: An essential resource providing expert advice on chemical tools for cancer research

Abstract Background: The Chemical Probes Portal (Portal) serves as a vital resource in the realm of cancer research and biomedical science generally - offering expert guidance on high-quality chemical tools, usually protein inhibitors, crucial for studying physiological and pathological processes and validating therapeutic targets. Researchers often grapple with selecting suitable chemical probes, inadvertently utilizing tools ill-suited for their purpose, and not following best practice, thereby contaminating the scientific literature and wasting valuable time and resources. To address this challenge, the Portal (https://www.chemicalprobes.org) was established as a non-profit, independent online platform, meticulously curating, annotating, and scoring the quality of chemical probes and providing associated information and advice. Results: The Portal stands apart as the sole expert-curated public resource evaluating the quality and utility of small-molecule chemical tool compounds for use in cancer research and wider biomedical research. More than 250 experts, comprising scientific leaders in chemical biology, medicinal chemistry, pharmacology, and related fields, contribute their experience and insights to assess featured chemical probes. Since 2021, a progressively updated and enhanced version of the Portal has been operational, housing over 1000 compounds targeting 500+ proteins across diverse families, now including PROTACs and molecular glues. We also flag 254 ‘Unsuitables’, which are compounds that should not be used to interrogate a specific protein target. Additionally, the Portal now offers informative pages, providing advice delineating guidelines for selecting well-validated compounds, including those suitable for rodent studies, necessitating specific pharmacokinetic and pharmacodynamic properties. Best practice use requires employing high-quality chemical probes from more than one distinct chemical class that target the same protein, incorporating an inactive control compound, and following the recommended concentration range - all aimed at mitigating off-target risks. Conclusion: The enhanced Chemical Probes Portal provides a vital platform for researchers to navigate and evaluate chemical probes, easily and effectively. It employs a rigorous expert review to ensure the quality of the probes listed. The resource not only aids high-quality probe selection but also provides advice through an Information Centre, offering guidelines, FAQs, and additional information on probe criteria and use. The continuous expansion and outreach efforts aim to make the Portal an indispensable tool for cancer researchers and the broader biomedical research community. Researchers are encouraged to participate in the Portal's development and contribute to the robustness of chemical probe use in scientific endeavors. Citation Format: Domenico Sanfelice, Albert Antolin, Alisa Crisp, Yi Chen, Paul Brennan, Susanne Müller, Bissan Al-Lazikani, Aled Edwards, Paul Workman. The Chemical Probes Portal: An essential resource providing expert advice on chemical tools for cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3542.

Abstract 3218: Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML

Abstract Background: Acute myeloid leukemia (AML) with KMT2A-rearrangements have a poor prognosis and so there is an urgent need for novel therapies. Menin inhibitors have shown promising anti-leukemic effects in KMT2A-rearranged and NPM1-mutated AML. However, the efficacy of menin inhibitors as single agents may be limited by the development of resistance mutations. Combining menin inhibitors with other epigenetic drugs may present a strategy to enhance their therapeutic potential. In this study, we performed a combination drug screen using an epigenetic drug library in KMT2A-rearranged (MV4;11) and NPM1-mutant (OCI-AML3) AML cell lines to identify synergistic therapies that can be used in conjunction with menin inhibitors. Methods: During assay optimization, MV4;11 and OCI-AML3 cells were treated with the menin inhibitor DSP-5336, and their viability was assessed. Quality control metrics were applied with Z’ values consistently greater than 0.5 for all assay plates and then the cells were subjected to a small molecule compound library screen containing a set of 932 epigenetic drugs. The screening process included a systematic evaluation of each drug's effect on cell viability in combination with the DSP-5336. Results: The combination drug screen demonstrated robust performance in both MV4;11 and OCI-AML3 cell lines, with reliable quality control metrics. Among the epigenetic drugs tested, five compounds were identified as potential hits that synergistically enhanced the efficacy of DSP-5336 across both cell lines.The five identified compounds exhibited enhanced anti-leukemic effects when combined with the menin inhibitor in either one of the cell lines. The targets of these drugs varied, including histone deacetylases, histone acetyltransferases and BRD4 inhibition, suggesting many alternate mechanisms of action involving epigenetic modulation. Remarkably, one of the 5 hits C301-3161, a KAT2A inhibitor, showed limited activity in either cell line alone but increased cell killing with DSP-5336 in both cell lines, highlighting its potential as a specific therapeutic for combining with DSP-5336 in AML patients. Conclusions: Our findings suggest combining menin inhibitors with other epigenetic drugs may improve AML treatment outcomes. The combination drug screen successfully identified four novel compounds that synergistically enhance menin inhibitor efficacy in both KMT2A-rearranged and NPM1-mutant AML cell lines. These compounds hold significant promise as potential therapeutic candidates for further investigation. Future studies should focus on elucidating the underlying mechanisms of action for each hit and evaluating their effects in preclinical animal models. Ultimately, this research may pave the way for the development of innovative combination therapeutic strategies for AML patients with KMT2A-rearragned and NPM1-mutant AML. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Skye Montoya, Maurizio Affer, Anya K. Sondhi, Alexandra Chirino, Claude-Henry Volmar, Shaun Brothers, Justin Watts, Justin Taylor. Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3218.

Abstract 7628: Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy

Abstract Metastatic disease drastically reduces clinical prognosis, and remains the primary cause of mortality from solid cancers, such as breast and pancreatic cancer. Since targeted therapeutic options are limited, we developed small molecule compounds to block activation (GTP loading) of the Rho GTPases Rac and Cdc42. Rac and Cdc42 are ideal molecular targets due to their regulation of cell migration, proliferation, invasion, and signaling in cancer and immunosuppressive cells in the tumor microenvironment. Our lead compound MBQ-167 is a dual Rac and Cdc42 inhibitor with IC50 of ~100 nM for inhibition of Rac/Cdc42 activation and the phosphorylation of their common downstream effector p21-activated kinase (PAK). MBQ-167 is highly effective in mouse models of breast cancer metastasis and is currently undergoing a Phase I clinical trial in advanced breast cancer patients. The purpose of this study is to investigate potential target molecules that can be used as biomarkers predictive of Rac/Cdc42 inhibitor efficacy. We recently published that in addition to inhibiting the growth and migration of breast and pancreatic cancer cells, MBQ-167 inhibits macrophage cell migration, immunosuppressive myeloid cell activation and release of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cancer. Transcriptomic data of tumors from mice treated with vehicle or MBQ-167 identified a number of immunomodulatory pathways downregulated by MBQ-167, including IL-6 and S100A family signaling, and Chitinase 3-like (CHI3L1/CHI4L1) genes coding for the human YKL-40 protein. We tested for the activation status of PAK (phospho (p)-PAK) as a diagnostic marker for Rac/Cdc42 inhibitor efficacy using breast cancer patient tissue in ex vivo culture treated with vehicle or MBQ-167 and found decreased p-PAK staining following immunohistochemistry of MBQ-167-treated tissue. From flow cytometry, using Alexa-488-tagged anti p-PAK antibody, we found that p-PAK was reduced from peripheral blood mononuclear cells (PBMCs) following MBQ-167. ELISA assays for YKL-40/CHI3L1 proteins after treatment of pancreatic cancer cell lines and macrophage-like cells with Rac/Cdc42 inhibitors show that MBQ-167 and the derivative MBQ-168 reduced CHI3L1 levels from conditioned media. These results demonstrate the potential of p-PAK and YKL-40/CHI3L1 from blood as biomarkers for Rac/Cdc42 inhibitor efficacy. This study impacts pharmacodynamic biomarker design for the planned Phase 2 clinical trials for MBQ-167 in breast and pancreatic cancer. Citation Format: Anamaris Torres-Sanchez, Ailed M. Cruz-Collazo, Michael Rivera-Robles, Olga Katsara, Stephanie Dorta-Estremera, Viviana Negron, Victor P. Carlo, Robert J. Schneider, Suranganie Dharmawardhane. Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7628.

Abstract 3307: c-Met targeting chaperone-mediated protein degrader for therapeutic intervention in NSCLC

Abstract The interaction between E3 ligases and chaperones is crucial for proteostasis, regulating more than 10% of total proteasomes and over 30% of human E3 ligases (Kevei, 2017). Chaperones also can recognize their substrates and direct them to degradation by the ubiquitin-proteasome system (UPS). Developing a targeted protein degradation (TPD) technology that utilizes these advantageous attributes of chaperones, termed chaperone-mediated protein degradation (CMPD) technology, would broaden the range of disease-causing target proteins and would efficiently degrade them by recruiting various intracellular E3 ligases through interaction with chaperones, compare to other TPD approaches. The c-mesenchymal-epithelial transition (c-MET) is recognized as primary cause of drug-resistance to EGFR inhibitors in non-small cell lung cancer (NSCLC). Herein, to degrade the c-MET, we synthesized bifunctional small molecule compound inducing c-MET and chaperones in proximity, termed CMPD-MET degrader, and evaluate it in MET-mutant NSCLC cell lines and mouse xenograft models. The efficacy and potency of CMPD-MET degrader were assessed using H596 and H1437 cells in vitro. Cell proliferation was measured using MTT assay and c-Met degradation assay was conducted, along with the examination of proteasome-dependent degradation via western blot. An in vivo H596 xenograft model gauged impact of CMPD-MET degrader via I.P. administration. In NSCLC with MET Exon 14 skipping mutation cell line H596 and H1437, treatment a OZD-MET degrader resulted inhibition of cell proliferation with IC50< 5 µM and subsequent proteasome-dependent c-MET degradation with DC50 ranging from 4-6 µM for 24h. The degradation of c-MET levels significantly induced by OZD-MET degrader, especially, the c-Met was degraded by 70.4% and 51.7% at 5 µM for 24h in H596 and H1437 cells, respectively. Also, OZ-MET degrader effectively suppressed c-MET mediated signaling and showed cleavage of PARP-1 in a dose-dependent manner. OZD-MET degrader induced selective degradation of c-MET showing at least more than about 5-fold selectivity over other chaperones-regulated client proteins.In the H596 xenograft model, OZD-MET degrader was administered in 5 dosage groups (minimum dose is 2.5 mg/kg) resulted in dramatic reduction in tumor size compared to the vehicle-treated group. Remarkably, administration of OZD-MET degrader revealed more than 90% degradation of c-Met in tumor tissues compared to the vehicle groups via western blots and fluorescence image. In contrast, c-MET inhibitor treated group showed no effect on degradation of c-MET in tumor tissues. In summary, we developed a novel c-Met targeting chaperone-mediated protein degrader, showed highly potent and selective proteasome-dependent c-MET degradation and notable inhibition of tumor size in xenograft models. These results provide a strong foundation for further evaluation in clinical trials. Citation Format: Han Jung Chae, Jisun Kim, SunHye Lee, Ho Yoo, Hyun-Kyoung Kim, Young-Jin Yu. c-Met targeting chaperone-mediated protein degrader for therapeutic intervention in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3307.