Abstract Background. Immunotherapies aimed at enhancing antitumor immune responses or delay immune cell exhaustion have shown encouraging results across multiple cancer types. However, immunotherapy still presents limited and variable response rates in immunosuppressive solid tumors, where cancer cells develop multiple strategies to evade and block antitumor immune responses. As a result, options to treat immunosuppressive tumors remain limited and this calls for the development of novel targeted immunotherapies to meet this critical need. Our group recently proved that an immunosuppressive pathway, named as the Cbl-b-Notch1 axis, exists in T cells and acts to constrain T cell function by means of increased Notch1 degradation by Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), following T cell activation. We also found that inhibiting Cbl-b using small-molecule inhibitors effectively restores Notch1 level and enhances CD8 and CD4 T cell functions in in vitro and ex vivo conditions. In this study, we aimed to demonstrate the effectiveness and the underlying mechanisms though which blocking the Cbl-b-Notch1 axis rewires the immunosuppressive tumor microenvironment to mount protective antitumor immune responses. Experimental procedures and results. This work leveraged a combination of genetic editing, targeted immune cell depletion, in silico, in vivo, ex vivo models and functional assays in primary cells. We found that blocking the Cbl-b-Notch1 axis, via Cbl-b inhibition, is effective in hindering tumor growth in immunosuppressive Triple-Negative Breast Cancer (TNBC) models (C0321 and 4T1) and pre-clinical tumor-derived organoids. We found that Cbl-b inhibition exerts its antitumor effects primarily by conferring a cytotoxic Th9 phenotype to CD4 T cells, via Notch1 stimulation which regulates cytotoxic and Th9 features, including Granzyme B (GZMB) and IL9 secretion. Cbl-b-inhibitor-primed CD4 T cells reshape the tumor microenvironment through multifaceted mechanisms: (i) exert direct cytotoxicity against tumor cells via GZMB, (ii) recruit and enable CD8 T cell cytotoxic responses, (iii) reprogram macrophages to produce Thrombospondin-1 (Thbs1), via Notch1-dependend release of IL9 in the tumor microenvironment and (iv) hence leads to strong anti-angiogenesis effects that cut off the nutrients supply within the tumor. These findings are consistent with TNBC patient data in which increased Notch1, IL9 and Thbs1 expression are predictive of a better prognosis and longer survival. Conclusions. Overall, our work identifies Cbl-b-Notch1 axis blockers, like Cbl-b inhibitors, as a promising strategy to reprogram anticancer immune responses, remodel the tumor microenvironment, and suppress tumor growth, providing a promising avenue for next-generation targeted immunotherapies. Citation Format: Zhi Huang, Mairah Khan, Laura Naldi, Brionna King, Luis Del Valle, Silvana Leit, Beth Browning, Christine Loh, Lucio Miele, Giulia Monticone. Cbl-b-Notch1 axis blockers rewire immunosuppressive tumors to drive cytotoxic and anti-angiogenic immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7782.

PGE2 plays important roles in immune cell function and in potentiating tissue regeneration. 15-PGDH is the key enzyme involved in inactivation of PGE2 and its inhibition therefore provides valuable therapeutic opportunity. We have solved the first cocrystal structure of 15-PGDH bound to small molecule inhibitors, enabling us to efficiently investigate and understand the key functionalities required for potency. Rational structure-based design coupled with a host of advanced computational methods, including FEP+ and WaterMap, were used to develop novel series of 15-PGDH inhibitors. Of note, a machine-learning (ML) model trained with potencies predicted by FEP+ yielded a powerful tool to guide synthetic priority across a large virtual chemical library. Ultimately, a lead compound demonstrated elevation of colonic PGE2 following IP administration in mice, consistent with our therapeutic hypothesis.

Recurrence, therapy resistance and metastasis remain major challenges in cancer therapy, accounting for nearly all deaths from cancer. The inability of current therapies to eliminate cancer stem cells (CSCs) is widely believed to underlie these challenges. Targeting molecular pathways that help maintain the CSCs could open new avenues to devise durable cancer therapies. The CBL-family E3 ubiquitin ligases (CBL, CBL-B and CBL-C) attenuate tyrosine kinase signaling through ubiquitin-dependent degradation of tyrosine kinase-coupled receptors and signaling components. Gene deletion studies have shown that CBL-B, the predominant family member expressed in mature immune cells, mediates tumor-induced “immune editing” to generate a pro-tumor immune microenvironment. This mechanism has provided one line of evidence to develop chemical CBL-B inhibitors as cancer immune modulators, and candidate inhibitors exhibit anti-tumor activity in mouse implant models, supporting the general idea that CBL-family E3s are potential therapeutic targets. Using mouse genetic models, we have established that CBL and CBL-B are redundantly required for the maintenance of adult stem cell compartments, and that this role is dependent on the core function of CBL/CBL-B as negative regulators of the AKT-mTOR axis downstream of tyrosine kinase signaling. As normal organ stem cell programs are frequently co-opted by CSCs; we used a 3-pronged approach to ask if CBL/CBL-B are required to maintain CSC function. Methods/Results: 1. Based on higher CBL/CBL-B expression in TNBC cell lines, and TCGA data that CBL/CBL-B mRNA expression positively correlates with shorter patient survival, we engineered Dox-inducible shRNAs in TNBC cell lines and showed that CBL/CBL-B KD impaired cell migration and invasion, reduced the population with CSC markers and impaired orthotopic xenograft tumorigenesis and metastatic seeding. 2. We generated a unique mouse model in which the C3(1)-TAg transgene drives mammary tumors known to resemble human triple-negative breast cancer (TNBC) and floxed-Cbl and Cblb genes can be inducibly deleted using tamoxifen (via CreERT2). Tumors of these mice established as tumor-derived organoids (TDOs) exhibited significant impairment of growth and CSC signature gene expression upon Cbl/Cblb KO. 3. Since the developed “CBL-B” inhibitors target a mechanism shared between CBL/CBL-B, we used two active compounds (NTX-801, NTX-944) and one inactive compound (NTX-616) developed by Nimbus Therapeutics. The active inhibitors mimicked the genetic Cbl/Cblb KO to inhibit the mouse mammary TDOs growth, while the inactive analog was without effect. Our results establish a tumor-intrinsic requirement of CBL/CBL-B to maintain breast cancer CSCs and add a critical second rationale for the use of chemical inhibitors of CBL/CBL-B as targeted therapeutics to counter BC recurrence and metastasis. Bhopal C. Mohapatra, Aaqib M. Bhat, Mohsin Raza, Haitao Luan, Santosh Shrestha, Sowmya Kolluru, Matthew D. Storck, Lusheng Li, Fu-Shan Kuo, Silvana Leit, Fang Qiu, Subodh M. Lele, David Ciccone, Christine Loh, Shibiao Wan, Vimla Band, Hamid Band. Targeting CBL and CBLB ubiquitin ligases to exhaust cancer stem cells in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5697.

Microsatellite instability (MSI-H) is a phenotypic consequence of defective mismatch repair (dMMR) in cancer and is observed in over 20 solid tumor types, with the highest prevalence in colorectal, gastric and endometrial cancers. While the treatment landscape of MSI-H is improving with the use of immune checkpoint inhibitors, substantial unmet medical need remains for patients with MSI-H cancers that fail to respond or relapse on standard-of-care therapies. Werner Syndrome Helicase (WRN) has been validated as a promising synthetic lethal drug target for MSI-H tumors, thus inhibitors of WRN may offer a novel therapeutic option for patients with MSI-H tumors. Our efforts to develop inhibitors of WRN led to the generation of two series of novel small molecules that had either a covalent or non-covalent mechanism of action. Treatment of MSI-H cell lines with exemplars from each series led to dose-dependent and rapid degradation of WRN protein, followed by an accelerated WRN protein re-synthesis rate that was independent of inhibitor mechanism. Washout studies using covalent inhibitors revealed that substantial target engagement was lost in MSI-H cells within 24- hours, suggesting rapid WRN resynthesis may limit sustained target inhibition. Prioritization of our non-covalent inhibitor series led to the generation of NTX-452, our lead candidate with best-in-class potential for the treatment of MSI-H cancers. The preclinical profile of non-covalent NTX-452 was characterized and compared to clinical-stage WRN inhibitors, including those from Novartis (HRO761, non-covalent) and Roche/Vividion (RO7589831, covalent). NTX-452 displayed excellent metabolic stability- with low in vivo clearance, good oral bioavailability, and high oral exposures in rodents and non-rodents. In vivo, low dose NTX-452 treatment induced robust pharmacodynamic and tumor regression across multiple MSI-H Cell line Derived (CDX) and Patient Derived (PDX) xenograft models. Notably, NTX-452 outperformed existing clinical stage WRN inhibitors at lower doses across multiple MSI-H tumor models. NTX-452 has broad efficacy across MSI-H CDX and PDX models from colorectal, gastric and endometrial lineages and is highly efficacious in models with diverse genomic and clinical characteristics. Moreover, NTX-452 promoted durable tumor regression and complete responses in MSI-H PDX models that were refractory to immunotherapy (anti-PD1) or chemotherapy. Lastly, resistance to clinical stage WRN inhibitors was explored and the potential for NTX-452 efficacy in WRN inhibitor resistant cell lines and tumors was evaluated.Taken together, our results highlight the broad, best-in-class potential of NTX-452 in MSI-H tumors and support its advancement to clinical evaluation. Rob Svensson, Scott Boiko, Beth Browning, Sam Carreiro, Derun Li, Angela Toms, Justin Caravella, Silvana Leit, Suzanne Jacques-O'Hagan, Hyesun Oh, Emily Florine, Zhenhong Li, Avinash Phadke, Yuet Mei Khong, Anita Scheuber, Peter Tummino. NTX-452: a non-covalent, potent, selective and highly efficacious WRN inhibitor with best-in-class potential for the treatment of MSI-H tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2894.

ADVERTISEMENT RETURN TO BOOKPREVChapterNEXTTARGETING SELECTIVE TYROSINE KINASE 2 (TYK2) INHIBITORS FOR THE TREATMENT OF AUTOIMMUNE DISEASESSilvana LeitSilvana LeitNimbus Therapeutics, Boston, Massachusetts, U.S.A.More by Silvana Leit, Bhaskar SrivastavaBhaskar SrivastavaNimbus Therapeutics, Boston, Massachusetts, U.S.A.More by Bhaskar Srivastava, Nathan E. GenungNathan E. GenungNimbus Therapeutics, Boston, Massachusetts, U.S.A.More by Nathan E. Genung, Joshua J. McElweeJoshua J. McElweeNimbus Therapeutics, Boston, Massachusetts, U.S.A.More by Joshua J. McElwee, Denise LevasseurDenise LevasseurNimbus Therapeutics, Boston, Massachusetts, U.S.A.More by Denise Levasseur, and Scott D. EdmondsonScott D. EdmondsonNimbus Therapeutics, Boston, Massachusetts, U.S.A.More by Scott D. EdmondsonDOI: 10.1021/mc-2023-vol58.ch07Publication Date (Web):November 16, 2023Publication History Published online16 November 2023Request reuse permissions Copyright © 2023 MEDI, Inc. Published by American Chemical Society.2023 Medicinal Chemistry ReviewsChapter 7pp 157-185Medicinal Chemistry ReviewsVol. 58ISBN13: 9781734427462eISBN: 9781734427462Article Views115Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (1 MB) Get e-Alerts

TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30, a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.

Introduction: Casitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase and a member of a highly conserved family of Cbl proteins, catalyzes the ubiquitination of substrate proteins to regulate multiple signaling events in a variety of cell types, including immune cells. In T cells, Cbl-b negatively regulates adaptive immune system signaling by establishing the threshold for the activation of antigen receptors. Additionally, Cbl-b regulates the function of other immune cell types, including NK cells, dendritic cells (DC) and monocytes. Cbl-b deficient T cells no longer require a costimulatory signal to be fully activated. Cbl-b KO mice spontaneously reject tumors via an enhanced immune response. Taken together, these findings point to Cbl-b inhibitors as having the potential to be highly efficacious immuno-oncology agents. Experimental Procedures: A structure-based drug design approach was used to identify potent inhibitors of Cbl-b. Biochemical and cellular binding assays, a cellular NFAT-luciferase reporter assay, as well as primary in vitro human and mouse T cell activation assays measuring IL-2 production were used to profile inhibitor compounds. In vivo activity of Cb-b inhibitors was evaluated using an anti-CD3 mouse model and a CT-26 syngeneic mouse model Results: Cbl-b inhibitor NTX-001 potently binds to Cbl-b, preventing Cbl-b phosphorylation and binding to E2. In cells, NTX-001 treatment results in enhanced phosphorylation of ZAP-70, a protein proximal to the TCR and reported to be a direct substrate of Cbl-b. Additionally, inhibition of Cbl-b potently enhances cytokine secretion from primary human and mouse T cells. In vivo, an increase in cytokines and T cell activation markers was observed after a single dose of NTX-001. Repeated dosing of NTX-001 showed dose-dependent anti-tumor activity in the colorectal CT-26 syngeneic model. Conclusions: NTX-001 is a potent Cbl-b inhibitor that demonstrated strong T cell activation and anti-tumor activity in a syngeneic tumor model. These data support Cbl-b inhibitors as a promising therapeutic opportunity for cancer treatment. Citation Format: Fred Csibi-Levin, Silvana Leit, David L. Laughton, Tom Baker, Suzanne L. Jacques, Beth Browning, Angela V. Toms, Samantha Garside, Simon D'Archivio, Katarzyna Kopycka, Xiaohua Zhu, Allan M. Jordan, Stuart Thomson, Christine Loh, Peter Tummino, David N. Ciccone. Discovery of NTX-001, a potent Cbl-b inhibitor with antitumor activity in syngeneic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 589.

BackgroundCasitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase and a member of a highly conserved family of Cbl proteins, catalyzes the ubiquitination of substrate proteins to regulate multiple signaling events in a variety of cell types, including immune cells. In T cells, Cbl-b negatively regulates adaptive immune system signaling by establishing the threshold for the activation of antigen receptors. Additionally, Cbl-b regulates the function of other immune cell types, including NK cells, dendritic cells (DC) and monocytes. Cbl-b deficient T cells no longer require a costimulatory signal to be fully activated. Cbl-b KO mice spontaneously reject tumors via an enhanced immune response. Taken together, these findings point to Cbl-b inhibitors as having the potential to be highly efficacious immuno-oncology agents.MethodsA structure-based drug design approach was used to identify potent inhibitors of Cbl-b. Biochemical and biophysical assays, in vitro cellular assays, as well as primary human and mouse immune cell assays assays were used to profile inhibitor compounds. In vivo activity of Cbl-b inhibitors was evaluated using an anti-CD3 mouse model and a CT-26 syngeneic mouse model.ResultsCbl-b inhibitors potently bind to Cbl-b, preventing Cbl-b phosphorylation and binding to E2. In cells, compound treatment results in enhanced transcriptional activity and robust cytokine secretion from primary human and mouse T cells. In vivo, an increase in cytokines and T cell activation markers was observed after a single dose of compound. Repeated dosing of compound showed dose-dependent anti-tumor activity in the colorectal CT-26 syngeneic model.ConclusionsPotent Cbl-b inhibitors demonstrate strong T cell activation and anti-tumor activity in a syngeneic tumor model. These data support Cbl-b inhibitors as a promising therapeutic opportunity for cancer treatment.

A critical feature of cancer is the ability to induce immunosuppression and evade immune responses. Tumor-induced immunosuppression diminishes the effectiveness of endogenous immune responses and decreases the efficacy of cancer immunotherapy. In this study, we describe a new immunosuppressive pathway in which adenosine promotes Casitas B-lineage lymphoma b (Cbl-b)-mediated Notch1 degradation, causing suppression of CD8+ T-cells effector functions. Genetic knockout and pharmacological inhibition of Cbl-b prevents Notch1 degradation in response to adenosine and reactivates its signaling. Reactivation of Notch1 results in enhanced CD8+ T-cell effector functions, anti-cancer response and resistance to immunosuppression. Our work provides evidence that targeting the Cbl-b-Notch1 axis is a novel promising strategy for cancer immunotherapy.

TYK2 is a member of the JAK family of kinases and a key mediator of IL-12, IL-23, and type I interferon signaling. These cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genetic association studies, TYK2 inhibition is an attractive therapeutic strategy for these diseases. Herein, we report the discovery of a series of highly selective catalytic site TYK2 inhibitors designed using FEP+ and structurally enabled design starting from a virtual screen hit. We highlight the structure-based optimization to identify a lead candidate 30, a potent cellular TYK2 inhibitor with excellent selectivity, pharmacokinetic properties, and in vivo efficacy in a mouse psoriasis model.

Background: Tumor-induced immunosuppression is a way in which cancers evade the host immune response. In recent years, a great effort has been made in designing immunotherapies that can boost the immune system response against cancer. However, not all tumors respond to these therapies. Therefore, there remains an unmet need for immunotherapies that are able to circumvent tumor-induced immunosuppression. Adenosine is an immunosuppressive metabolite which is overproduced in hypoxic tumor microenvironments and dampens T-cell anti-tumor immune responses. Adenosine A2A receptor (A2AR) activation was shown to downregulate Notch1, a key regulator of T-cell functions, in CD8+ T-cells, leading to immunosuppression. Notch1 signaling appears to protect T-cells against immunosuppressive signals, as Notch1 overexpressing T-cells were shown to be resistant to tumor-induced immunosuppression mediated by myeloid-derived suppressor cells (MDSC), as well as adenosine. We hypothesize that rescuing Notch1 from downregulation will make T-cells resistant to tumor-induced immunosuppression. We applied several functional assays in primary CD8+ T-cells and tumor-derived organoids to study how Notch1 is regulated by A2AR and explore strategies to target this pathway for cancer immunotherapy. Results: Our results indicate that the ubiquitin ligase Cbl-b, a negative regulator of T-cell functions, is responsible for the ubiquitination and degradation of Notch1 in CD8+ T-cells. A2AR signaling leads to Notch1 downregulation by promoting Cbl-b-mediated Notch1 degradation. Inhibition of Cbl-b using small molecule compounds restores Notch1 and T-cell functions in CD8+ T-cells in the presence of adenosine, making them resistant to A2AR-mediated immunosuppression. Cbl-b inhibitors show anti-tumor activity in tumor-derived organoids from pre-clinical models and enhance immune-checkpoint immunotherapy, by promoting T-cells anti-cancer responses. Conclusions: Our work suggests that promoting Notch1 signaling by blocking Cbl-b-mediated degradation results in increased T-cell responses and resistance to immunosuppression. Targeting Cbl-b-Notch1 axis represents a promising novel strategy to boost anti-cancer T-cell responses. Citation Format: Giulia Monticone, Fred Csibi, Silvana Leit, Jermaine E. Austin, Deniz A. Ucar, Fokhrul M. Hossain, Samarpan Majumder, Barbara A. Osborne, Christine Loh, Lucio Miele. Targeting Cblb-Notch1 axis as a novel strategy for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2064.

Aim Type 1 diabetes (T1D) is a chronic autoimmune disease leading to progressive loss of pancreatic beta cells. Interferon (IFN)-alpha plays a critical role in the crosstalk between pancreatic beta cells and the immune system in early insulitis. In human beta cells IFN alpha signals through JAK1 and TYK2, leading to endoplasmic reticulum stress, inflammation and HLA class I overexpression. IFN alpha, acting synergistically with IL-1 beta, induces apoptosis. Polymorphisms in TYK2 that decrease its activity are associated with protection against T1D, and we hypothesized that pharmacological inhibitors that specifically target TYK2 could protect human beta cells against the deleterious effects of IFN alpha. Materials and Methods Two TYK2 inhibitors provided by Nimbus Lakshmi were tested in human insulin-producing EndoC-beta H1 cells and human islets to evaluate their effect on IFN alpha signalling, beta-cell function and susceptibility to viral infection using RT-qPCR, western blot, immunofluorescence, ELISA and nuclear dyes. Results The two TYK2 inhibitors tested prevented IFN alpha-induced human beta-cell gene expression in a dose-dependent manner. They also protected human islets against IFN alpha + IL-1 beta-induced apoptosis. Importantly, these inhibitors did not modify beta-cell function or their survival following infection with the potential diabetogenic coxsackieviruses CVB1 and CVB5. Conclusions The two TYK2 inhibitors tested inhibit the IFN alpha signalling pathway in human beta cells, decreasing its pro-inflammatory and pro-apoptotic effects without sensitizing the cells to viral infection. The preclinical findings could pave the way for future clinical trials with TYK2 inhibitors for the prevention and treatment of type 1 diabetes.

ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA1-5. The acetyl-CoA product is crucial for the metabolism of fatty acids6,7, the biosynthesis of cholesterol8, and the acetylation and prenylation of proteins9,10. There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation2,5,11. ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials4,5. Many inhibitors of ACLY have been reported, but most of them have weak activity5. Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have also determined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure-activity relationships of these compounds. This allosteric site greatly enhances the 'druggability' of ACLY and represents an attractive target for the development of new ACLY inhibitors.

In an effort to identify HDAC isoform selective inhibitors, we designed and synthesized novel, chiral 3,4-dihydroquinoxalin-2(1H)-one and piperazine-2,5-dione aryl hydroxamates showing selectivity (up to 40-fold) for human HDAC6 over other class I/IIa HDACs. The observed selectivity and potency (IC(50) values 10-200 nM against HDAC6) is markedly dependent on the absolute configuration of the chiral moiety, and suggests new possibilities for use of chiral compounds in selective HDAC isoform inhibition.

The sulfamide moiety has been utilized to design novel HDAC inhibitors. The potency and selectivity of these inhibitors were influenced both by the nature of the scaffold, and the capping group. Linear long-chain-based analogs were primarily HDAC6-selective, while analogs based on the lysine scaffold resulted in potent HDAC1 and HDAC6 inhibitors.

We have identified a series of diphenylmethylene hydroxamic acids as novel and selective HDAC class IIa inhibitors. The original hit, N-hydroxy-2,2-diphenylacetamide (6), has sub-micromolar class IIa HDAC inhibitory activity, while the rigidified oxygen analogue, N-hydroxy-9H-xanthene-9-carboxamide (13), is slightly more selective for HDAC7 with an IC(50) of 0.05muM. Substitution of 6 allows for the modulation of selectivity and potency amongst the class IIa HDAC isotypes.