Small-molecule Binding Pockets Research Articles

Integrating Large-Scale Protein Structure Prediction into Human Genetics Research.

The last five years have seen impressive progress in deep learning models applied to protein research. Most notably, sequence-based structure predictions have seen transformative gains in the form of AlphaFold2 and related approaches. Millions of missense protein variants in the human population lack annotations, and these computational methods are a valuable means to prioritize variants for further analysis. Here, we review the recent progress in deep learning models applied to the prediction of protein structure and protein variants, with particular emphasis on their implications for human genetics and health. Improved prediction of protein structures facilitates annotations of the impact of variants on protein stability, protein-protein interaction interfaces, and small-molecule binding pockets. Moreover, it contributes to the study of host-pathogen interactions and the characterization of protein function. As genome sequencing in large cohorts becomes increasingly prevalent, we believe that better integration of state-of-the-art protein informatics technologies into human genetics research is of paramount importance.

Predicted mechanistic impacts of human protein missense variants.

Genome sequencing efforts have led to the discovery of tens of millions of protein missense variants found in the human population with the majority of these having no annotated role and some likely contributing to trait variation and disease. Sequence-based artificial intelligence approaches have become highly accurate at predicting variants that are detrimental to the function of proteins but they do not inform on mechanisms of disruption. Here we combined sequence and structure-based methods to perform proteome-wide prediction of deleterious variants with information on their impact on protein stability, protein-protein interactions and small-molecule binding pockets. AlphaFold2 structures were used to predict approximately 100,000 small-molecule binding pockets and stability changes for over 200 million variants. To inform on protein-protein interfaces we used AlphaFold2 to predict structures for nearly 500,000 protein complexes. We illustrate the value of mechanism-aware variant effect predictions to study the relation between protein stability and abundance and the structural properties of interfaces underlying trans protein quantitative trait loci (pQTLs). We characterised the distribution of mechanistic impacts of protein variants found in patients and experimentally studied example disease linked variants in FGFR1.

Decreasing the intrinsically disordered protein α-synuclein levels by targeting its structured mRNA with a ribonuclease-targeting chimera.

α-Synuclein is an important drug target for the treatment of Parkinson's disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5' untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5' UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient-derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.

Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics

The nucleus controls cell growth and division through coordinated interactions between nuclear proteins and chromatin. Mutations that impair nuclear protein association with chromatin are implicated in numerous diseases. Covalent ligands are a promising strategy to pharmacologically target nuclear proteins, such as transcription factors, which lack ordered small-molecule binding pockets. To identify nuclear cysteines that are susceptible to covalent liganding, we couple proximity labeling (PL), using a histone H3.3-TurboID (His-TID) construct, with chemoproteomics. Using covalent scout fragments, KB02 and KB05, we identified ligandable cysteines on proteins involved in spindle assembly, DNA repair, and transcriptional regulation, such as Cys101 of histone acetyltransferase 1 (HAT1). Furthermore, we show that covalent fragments can affect the abundance, localization, and chromatin association of nuclear proteins. Notably, the Parkinson disease protein 7 (PARK7) showed increased nuclear localization and chromatin association upon KB02 modification at Cys106. Together, this platform provides insights into targeting nuclear cysteines with covalent ligands.

Virus-like particle-based delivery of Cas9/guide RNA ribonucleoprotein efficiently edits the brachyury gene and inhibits chordoma growth in vivo

PurposeChordoma is a rare and aggressive bone cancer driven by the developmental transcription factor brachyury. Efforts to target brachyury are hampered by the absence of ligand-accessible small-molecule binding pockets. Genome editing with CRISPR systems provides an unprecedented opportunity to modulate undruggable transcription factor targets. However, delivery of CRISPR remains a bottleneck for in vivo therapy development. The aim was to investigate the in vivo therapeutic efficiency of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) delivery through a novel virus-like particle (VLP) by fusing an aptamer-binding protein to the lentiviral nucleocapsid protein.MethodsThe p24 based ELISA and transmission electron microscopy were used to determine the characterization of engineered VLP-packaged Cas9/gRNA RNP. The deletion efficiency of brachyury gene in chordoma cells and tissues was measured by genome cleavage detection assay. RT-PCR, Western blot, immunofluorescence staining, and IHC were employed to test the function of brachyury deletion. Cell growth and tumor volume were measured to evaluate the therapeutic efficiency of brachyury deletion by VLP-packaged Cas9/gRNA RNP.ResultsOur “all-in-one” VLP-based Cas9/gRNA RNP system allows for transient expression of Cas9 in chordoma cells, but maintains efficient editing capacity leading to approximately 85% knockdown of brachyury with subsequent inhibition of chordoma cell proliferation and tumor progression. In addition, this VLP-packaged brachyury-targeting Cas9 RNP avoids systemic toxicities in vivo.ConclusionOur preclinical studies demonstrate the potential of VLP-based Cas9/gRNA RNP gene therapy for the treatment of brachyury-dependent chordoma.

PO-01-178 MAPPING OF PATHOGENIC VARIANT HOTSPOTS ON THE STRUCTURE OF RYANODINE RECEPTOR TYPE 2 DRIVES A RATIONAL DRUG DESIGN APPROACH FOR CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA

Heritable mutations in RYR2 cause sudden-death predisposing arrhythmia syndromes, such as catecholaminergic polymorphic ventricular tachycardia (CPVT), through increased sarcoplasmic reticulum (SR) diastolic Ca2+ leak. RYR2-encoded ryanodine receptor 2 is an intracellular transmembrane Ca2+ release channel in the SR of cardiomyocytes. Even on optimal medical therapy, breakthrough life-threatening arrhythmias in patients with CPVT are common. We sought to correlate identified pathogenic hotspots with key functional domains on the 3D structure of RyR2 and possible mobile binding sites to determine small molecule targets for reducing SR Ca2+ leak. We performed “signal” to “noise” (S:N) analysis in RYR2 to establish pathogenic hotspots. The minor allele frequency (MAF) of literature-derived disease-associated genetic variants at a given amino acid locus was normalized to the MAF of rare population-based variants in Genome Aggregation Database (gnomAD). Using PyMOL, a heat map overlay was generated using S:N levels and possible molecular binding pockets were identified using software PyMOL and Maestro. The highest cumulative S:N intensity of pathogenic hotspots of each domain localized on the channel domain (28.2%), helical domain 1 (25.6%), central domain (17.7%), and N-terminal domain (16.5%) of RyR2. The SPRY1 (0.498%), P1 (0.147%), handle domain (2.32%), and central U motif (8.88%) had lower S:N. Cumulative S:N intensities were nonexistent for SPRY2, SPRY3, P2, and helical domain 2. Putative small molecule binding pockets were identified in all domains while the largest number localized to the channel domain. Once binding pockets were combined with the heat map structure of RyR2, activator binding pockets of low S:N threshold in the open state of RyR2 were shown to be located heavily on the P2 domain, where CaMKII and PKA binds. The highest S:N intensity was in the channel domain with high S:N levels in other proximal domains localizing near the SR transmembrane in the lumen. Mobile binding pockets between the open and closed state of RyR2 with low S:N intensity may be alternative areas for drug targeting, especially on domains that contain binding pockets for activators of RYR2.

NanoBeacon.AI: AI-Enhanced Nanodiamond Biosensor for Automated Sensitivity Prediction to Oxidative Phosphorylation Inhibitors.

Spalt-like transcription factor 4 (SALL4) is an oncofetal protein that has been identified to drive cancer progression in hepatocellular carcinoma (HCC) and hematological malignancies. Furthermore, a high SALL4 expression level is correlated to poor prognosis in these cancers. However, SALL4 lacks well-structured small-molecule binding pockets, making it difficult to design targeted inhibitors. SALL4-induced expression of oxidative phosphorylation (OXPHOS) genes may serve as a therapeutically targetable vulnerability in HCC through OXPHOS inhibition. Because OXPHOS functions through a set of genes with intertumoral heterogeneous expression, identifying therapeutic sensitivity to OXPHOS inhibitors may not rely on a single clear biomarker. Here, we developed a workflow that utilized molecular beacons, nucleic-acid-based, activatable sensors with high specificity to the target mRNA, delivered by nanodiamonds, to establish an artificial intelligence (AI)-assisted platform for rapid evaluation of patient-specific drug sensitivity. Specifically, when the HCC cells were treated with the nanodiamond-medicated OXPHOS biosensor, high sensitivity and specificity of the sensor allowed for improved identification of OXPHOS expression in cells. Assisted by a trained convolutional neural network, drug sensitivity of cells toward an OXPHOS inhibitor, IACS-010759, could be accurately predicted. AI-assisted OXPHOS drug sensitivity assessment could be accomplished within 1 day, enabling rapid and efficient clinical decision support for HCC treatment. The work proposed here serves as a foundation for the patient-based subtype-specific therapeutic research platform and is well suited for precision medicine.

Proof-of-Concept Method to Study Uncharacterized Methyltransferases Using PRDM15.

The PRDM family of methyltransferases has been implicated in cellular proliferation and differentiation and is deregulated in human diseases, most notably in cancer. PRDMs are related to the SET domain family of methyltransferases; however, from the 19 PRDMs only a few PRDMs with defined enzymatic activities are known. PRDM15 is an uncharacterized transcriptional regulator, with significant structural disorder and lack of defined small-molecule binding pockets. Many aspects of PRDM15 are yet unknown, including its structure, substrates, reaction mechanism, and its methylation profile. Here, we employ a series of computational approaches for an exploratory investigation of its potential substrates and reaction mechanism. Using the knowledge of PRDM9 and current knowledge of PRDM15 as basis, we tried to identify genuine substrates of PRDM15. We start from histone-based peptides and learn that the native substrates of PRDM15 may be non-histone proteins. In the future, a combination of sequence-based approaches and signature motif analysis may provide new leads. In summary, our results provide new information about the uncharacterized methyltransferase, PRDM15.

Opportunities and challenges of protein-based targeted protein degradation.

In the 20 years since the first report of a proteolysis targeting chimeric (PROTAC) molecule, targeted protein degradation (TPD) technologies have attempted to revolutionize the fields of chemical biology and biomedicine by providing exciting research opportunities and potential therapeutics. However, they primarily focus on the use of small molecules to recruit the ubiquitin proteasome system to mediate target protein degradation. This then limits protein targets to cytosolic domains with accessible and suitable small molecule binding pockets. In recent years, biologics such as proteins and nucleic acids have instead been used as binders for targeting proteins, thereby expanding the scope of TPD platforms to include secreted proteins, transmembrane proteins, and soluble but highly disordered intracellular proteins. This perspective summarizes the recent TPD platforms that utilize nanobodies, antibodies, and other proteins as binding moieties to deplete challenging targets, either through the ubiquitin proteasome system or the lysosomal degradation pathway. Importantly, the perspective also highlights opportunities and remaining challenges of current protein-based TPD technologies.

Abstract 1192: Mapping small molecule binding pockets on lipid kinases using chemoproteomics and AlphaFold

Abstract 1192: Mapping small molecule binding pockets on lipid kinases using chemoproteomics and AlphaFold

Predicting small molecule binding pockets on diacylglycerol kinases using chemoproteomics and AlphaFold.

Diacylglycerol kinases (DGKs) are metabolic kinases involved in regulating cellular levels of diacylglycerol and phosphatidic lipid messengers. The development of selective inhibitors for individual DGKs would benefit from discovery of protein pockets available for inhibitor binding in cellular environments. Here we utilized a sulfonyl-triazole probe (TH211) bearing a DGK fragment ligand for covalent binding to tyrosine and lysine sites on DGKs in cells that map to predicted small molecule binding pockets in AlphaFold structures. We apply this chemoproteomics-AlphaFold approach to evaluate probe binding of DGK chimera proteins engineered to exchange regulatory C1 domains between DGK subtypes (DGKα and DGKζ). Specifically, we discovered loss of TH211 binding to a predicted pocket in the catalytic domain when C1 domains on DGKα were exchanged that correlated with impaired biochemical activity as measured by a DAG phosphorylation assay. Collectively, we provide a family-wide assessment of accessible sites for covalent targeting that combined with AlphaFold revealed predicted small molecule binding pockets for guiding future inhibitor development of the DGK superfamily.

Resolving neuroinflammation in AD

AbstractBackgroundHuman genetics point towards a key role of microglia and microglia expressed genes like TREM2 in neuroinflammation and AD pathology, yet our understanding of microglia spatial and temporal dynamics, therapeutic directionality and translational biomarkers remains limited.Method and ResultsMuna Therapeutics applies structural biology, computational chemistry and human IPSC derived cell assays to develop small molecule agonists for microglial drug targets including TREM2. Introduction of human mutations allows specific drug – target interactions to be probed and has revealed novel small molecule binding pockets, enabling optimized drug design. To identify novel drug targets involved in neuroinflammation, we have built an all‐in‐human spatial transcriptomics and bioinformatics‐based discovery and validation platform. We have assessed >50 human brain samples for pathology‐induced changes in gene expression in microglia and other cells from samples with AD pathology from patients with and without cognitive impairment. This approach has revealed patterns of differentially expressed genes and pathways in brains linked to pathological hallmarks such as Ab plaques and tau tangles, as well as to resilience to the effects of these pathologies. Candidate targets and mechanisms are tested for their impact on neuroinflammation, including directionality, in humanized model systems that also enable translational biomarker discovery and validation.ConclusionAt Muna Tx, state‐of‐the‐art target discovery and validation as well as structural biology and computational chemistry platforms enable microglia targeted drug discovery and can accelerate therapeutic advancement to clinical development.

Abstract B37: PLX-4107, a selective IKZF2 degrader, reprograms suppressive regulatory T cells and demonstrates anti-tumor activity

Abstract Immune checkpoint therapy has demonstrated durable clinical responses in multiple solid tumor types. Reduced clinical response to checkpoint therapy has been linked to the presence of potent immunosuppressive regulatory T cells (Tregs) within the tumor microenvironment that contribute to tumor immune evasion. The transcription factor Helios (IKZF2) is a marker of highly suppressive Tregs and is required to maintain a stable, suppressive Treg cell phenotype in the inflammatory tumor microenvironment. Depletion of IKZF2 in Tregs results in both loss of suppressive activity and conversion of Tregs into effector-like T cells, leading to anti-tumor immunity. Targeted protein degradation using the endogenous Ubiquitin Proteasome System (UPS) has enabled targeting undruggable proteins, such as IKZF2, that have no known small molecule binding pocket. We have designed small molecules that promote a novel interaction between IKZF2 with the E3 ubiquitin ligase substrate receptor, Cereblon, leading to proximity induced protein degradation. PLX-4107 is a novel molecular glue that is a highly selective, potent, and rapid degrader of IKZF2 via the redirection of the E3 substrate receptor, Cereblon. Degradation of IKZF2 by PLX-4107 is blocked in the presence of proteasome and neddylation inhibitors as well as a Cereblon knock-out cell line, confirming that degradation is mediated by the UPS and specifically through the involvement of Cereblon. Proteome-wide analysis demonstrated that PLX-4107 selectively depletes IKZF2 protein levels without degrading other known Cereblon neo-substrates. In vitro, PLX-4107 mediated degradation of IKZF2 resulted in conversion of suppressive Tregs into CD4+ effector-like T cells, coupled with an increased production of the effector cytokines IL2 and IFNg. Oral administration of PLX-4107 to cynomolgus monkeys demonstrated sustained pharmacodynamic response, persistent depletion of IKZF2, and reprogramming of Tregs, consistent with the catalytic mechanism of protein degradation. In vivo, T cell expansion studies showed that administration of PLX-4107 decreased both Treg CD25 expression and proliferation, along with increased activation of CD8+ T effector cells. PLX-4107 was evaluated in in vivo xenograft efficacy studies and demonstrated dose dependent single agent anti-tumor activity that was dependent on the presence of T cells. In addition, co-administration of PLX-4107 and anti-PD-1 antibody Pembrolizumab resulted in tumor growth inhibition and significant combination benefit. PLX-4107 is a novel molecular glue that selectively degrades the undruggable transcription factor, IKZF2. PLX-4107 mediated IKZF2 degradation results in conversion of Tregs to an effector-like T cell phenotype, single agent antitumor activity and the ability to enhance the efficacy of immune checkpoint therapy in vivo. Citation Format: Peggy A Thompson, Pengyu Yang, Linette Yang, Susan Song, Yujun Huang, Xiaoming Li, Alejandro Dearie, Stephen Chien, Mary E Spalding, Gabrielle Blanco, Elizabeth Daniele, Julia Toth, Aleksandar Jamborcic, Gregory Parker, Simon Bailey. PLX-4107, a selective IKZF2 degrader, reprograms suppressive regulatory T cells and demonstrates anti-tumor activity [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B37.

EXTH-55. DELIVERY OF CAS9/SGRNA RIBONUCLEOPROTEIN CONJUGATE VIA VIRUS-LIKE PARTICLES FOR EFFICIENT GENE EDITING OF TRANSCRIPTION FACTOR BRACHYURY IN GLIOBLASTOMA

Abstract Brachyury, a transcription factor that is a critical driver during normal embryonic development, is highly expressed in glioblastoma (GBM) but rarely expressed in normal adult tissues. Recent evidence suggests that brachyury plays a crucial role in regulating GBM cell proliferation and stemness, implying the potential of brachyury as a therapeutic target. However, like other transcription factors, brachyury is not readily inhibited pharmacologically due to the absence of ligand-accessible small-molecule binding pockets, and a direct inhibitor of brachyury has not been identified. Our team has recently developed a novel virus-like particle (VLP)-based system by fusing aptamer-binding protein to the lentiviral nucleocapsid protein within the group-specific antigen, which allows for efficient packaging of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) for gene editing. Our data suggests that our VLP-mediated Cas9/gRNA RNP allows for transient expression of Cas9 in patient-derived GBM cells while maintaining effective gene editing efficiency (∼40%). Our strategy results in 90% knockdown of brachyury in vitro with subsequent inhibition of GBM cell proliferation and stemness as well as enhanced susceptibility of tumor cells to radiation via increased cleavage of caspase-3. In addition, our in vivo results using a xenograft mouse model further demonstrates that transduction with VLP-packaged Cas9/brachyury gRNA inhibits GBM progression. Our findings demonstrate that our novel VLP-based system allows for efficient delivery of Cas9/gRNA RNP-based brachyury gene editing technology and represents a novel treatment strategy for GBM.

Two Binding Sites of SARS-CoV-2 Macrodomain 3 Probed by Oxaprozin and Meclomen.

Severe acute respiratory syndrome-coronavirus-1/2 (SARS-CoV-1/2) macrodomain 3 (Mac3) is critical for replication and transcription of the viral genome and is therefore a potential therapeutic target. Here, we solved the crystal structure of SARS-CoV-2 Mac3, which reveals a small-molecule binding pocket. Two low-molecular-weight drugs, oxaprozin and meclomen, induced different patterns of nuclear magnetic resonance (NMR) chemical shift perturbations (CSPs). Meclomen binds to site I of SARS-CoV-2 Mac3 with binding pose determined by NMR CSP and transferred paramagnetic relaxation enhancement, while oxaprozin binds to site II as revealed by the crystal structure. Interestingly, oxaprozin and meclomen both perturb residues in site I of SARS-CoV Mac3. Fluorescence polarization experiments further demonstrated that oxaprozin and meclomen inhibited the binding of DNA-G4s to SARS-CoV-2 Mac3. Our work identified two adjacent ligand-binding sites of SARS-CoV-2 Mac3 that shall facilitate structure-guided fragment linking of these compounds for more potent inhibitors.

Abstract 1111: Identification of selective IKZF2 degraders that reprogram suppressive regulatory T cells in solid tumors

Abstract Targeted protein degradation (TPD) using the endogenous Ubiquitin Proteasome System (UPS) is a rapidly growing drug discovery strategy to eliminate pathogenic proteins. Molecular glues are small molecules that promote a novel interaction between a protein of interest with an E3 ubiquitin ligase leading to proximity induced protein degradation. This has enabled targeting undruggable proteins, such as the zinc-finger transcription factor Helios (IKZF2), that have no known small molecule binding pocket. Despite recent clinical breakthroughs in checkpoint blockade in treating solid tumors, suppression of the antitumor immune response in the tumor microenvironment (TME) is a major obstacle to tumor regression. Regulatory T cells (Tregs) in the TME are potent immunosuppressive cells that promote progression of cancer. IKZF2 has been shown to be a marker of highly suppressive Treg cells and is critical for maintaining the anergic and suppressive phenotype in the highly inflammatory tumor microenvironment. Genetic depletion of IKZF2 in Treg cells results in both loss of suppressive activity and conversion of Tregs into T effector cells, leading to enhanced anti-tumor immunity. Collectively, these findings support that an IKZF2-specific degrader could be beneficial in enhancing the efficacy of current immunotherapies. Here we report our efforts leading to the development of a series of potent and selective IKZF2 degraders for the treatment of cancer. Our lead compound, PLX-4107, is a novel molecular glue that was optimized to be a highly selective, deep, and rapid IKZF2 degrader via the redirection of the E3 substrate receptor, cereblon. Proteome-wide analysis demonstrated that PLX-4107 depletes IKZF2 protein levels without degrading other known cereblon neo-substrates. PLX-4107 mediated degradation of IKZF2 resulted in conversion of highly suppressive Tregs into T effector cells, coupled with an increased expression of the effector cytokines IL2 and IFNγ. Oral administration of PLX-4107 to cynomolgus monkeys caused rapid, complete, and prolonged degradation of IKZF2 in Tregs, indicative of the catalytic nature of degraders where sustained pharmacodynamic response is observed well beyond plasma drug exposure levels. These preliminary data provide a strong rationale for developing small molecule therapeutics that target the undruggable IKZF2 transcription factor with the potential to enhance the efficacy of immune checkpoint therapy. Citation Format: Peggy A. Thompson, Pengyu Yang, Xiaoming Li, Stephen Chien, Mary E. Spalding, Alejandro Dearie, Elizabeth Daniele, Linette Yang, E Adam Kallel, Aleksandar Jamborcic, Julia Toth, Gregory Parker, Simon Bailey. Identification of selective IKZF2 degraders that reprogram suppressive regulatory T cells in solid tumors [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 1111.

Abstract 429: Androgen receptor (AR) N-terminal domain degraders can degrade AR full length and AR splice variants in CRPC preclinical models

Abstract Introduction: Androgen receptor (AR) signaling is a main driver of prostate cancer progression and remains a crucial target for therapeutic intervention even in late stages of the disease. Current antiandrogen therapies directly or indirectly target the AR ligand binding domain (LBD) and are initially effective in prostate cancer patients. However, resistance ultimately develops, and new methods of inhibiting the AR pathway are needed. The selective targeting of the N-terminal domain (NTD) of the AR by small molecule anitens represents a novel method of blocking AR signaling that can bypass LBD-related resistance mechanisms. AR NTD is an intrinsically disordered region (IDR) which has no stable ordered structure and is generally regarded as an undruggable target due to the lack of a well-defined small-molecule binding pocket. However, we recently demonstrated that EPI-7386, an AR NTD small molecule inhibitor, inhibits AR activity by binding to Tau5 region of the NTD. By developing an aniten based bifunctional degrader (ANITAC for ANITen bAsed Chimera), our goal is to eliminate any forms of AR protein found in castration-resistant prostate cancer (CRPC), that can potentially drive disease progression, including LBD mutants and AR truncated variants. Methods: AR degradation was monitored by Western Blot or by using the HiBit assay. AR transcriptional activity was measured using different reporter assays following AR full length (FL) or truncated AR activity in in vitro models. Results: Here we report the first series of AR degraders targeting the NTD of AR, which lead to degradation of all forms of AR, including AR-V7 and AR-v567es. ANITACs eliminate AR in all cell lines tested through an E3 ligase dependent mechanism, with an observed 50% degradation concentration (DC50) < 20 nM in 22Rv1 cells for the most potent compounds. AR degradation mediated by ANITACs suppresses the expression of AR target genes and decreases the viability of prostate cancer cells. ANITACs also degrade clinically relevant AR mutants and AR splice variants and show inhibition of AR transcriptional activity in multiple cell lines expressing different forms of AR including AR FL (LNCaP, CWR-AD1), AR-V7 (22Rv1), AR-V567es (CWR-D567). In vitro and in vivo PK studies show the compounds are metabolically stable and exhibit excellent oral bioavailability in mice. Conclusion: In summary, we report preclinical data on the first generation of ANITAC molecules, that can be orally bioavailable and show activity against forms of AR expressed in late stage CRPC patients. Citation Format: Nan Hyung Hong, Berenger Biannic, Peter Virsik, Han-Jie Zhou, Ronan Le Moigne. Androgen receptor (AR) N-terminal domain degraders can degrade AR full length and AR splice variants in CRPC preclinical 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 429.

Cell-Based Drug Discovery: Identification and Optimization of Small Molecules that Reduce c-MYC Protein Levels in Cells.

Elevated expression of the c-MYC oncogene is one of the most common abnormalities in human cancers. Unfortunately, efforts to identify pharmacological inhibitors that directly target MYC have not yet yielded a drug-like molecule due to the lack of any known small molecule binding pocket in the protein, which could be exploited to disrupt MYC function. We have recently described a strategy to target MYC indirectly, where a screening effort designed to identify compounds that can rapidly decrease endogenous c-MYC protein levels in a MYC-amplified cell line led to the discovery of a compound series that phenocopies c-MYC knockdown by siRNA. Herein, we describe our medicinal chemistry program that led to the discovery of potent, orally bioavailable c-MYC-reducing compounds. The development of a minimum pharmacophore model based on empirical structure activity relationship as well as the property-based approach used to modulate pharmacokinetics properties will be highlighted.

Engineering Bioactive Dimeric Transcription Factor Analogs via Palladium Rebound Reagents.

Transcription factors (TF), such as Myc, are proteins implicated in disease pathogenesis, with dysregulation of Myc expression in 50% of all human cancers. Still, targeting Myc remains a challenge due to the lack of small molecule binding pockets in the tertiary structure. Here, we report synthetic covalently linked TF mimetics that inhibit oncogenic Myc-driven transcription by antagonistic binding of the target DNA-binding site. We combined automated flow peptide chemistry with palladium(II) oxidative addition complexes (OACs) to engineer covalent protein dimers derived from the DNA-binding domains of Myc, Max, and Omomyc TF analogs. Palladium-mediated cross-coupling of synthesized protein monomers resulted in milligram quantities of seven different covalent homo- and heterodimers. The covalent helical dimers were found to bind DNA and exhibited improved thermal stability. Cell-based studies revealed the Max-Max covalent dimer is cell-penetrating and interfered with Myc-dependent gene transcription resulting in reduced cancer cell proliferation (EC50 of 6 μM in HeLa). RNA sequencing and gene analysis of extracted RNA from treated cancer cells confirmed that the covalent Max-Max homodimer interferes with Myc-dependent transcription. Flow chemistry, combined with palladium(II) OACs, has enabled a practical strategy to generate new bioactive compounds to inhibit tumor cell proliferation.

Advances in targeting 'undruggable' transcription factors with small molecules.

Transcription factors (TFs) represent key biological players in diseases including cancer, autoimmunity, diabetes and cardiovascular disease. However, outside nuclear receptors, TFs have traditionally been considered 'undruggable' by small-molecule ligands due to significant structural disorder and lack of defined small-molecule binding pockets. Renewed interest in the field has been ignited by significant progress in chemical biology approaches to ligand discovery and optimization, especially the advent of targeted protein degradation approaches, along with increasing appreciation of the critical role a limited number of collaborators play in the regulation of key TF effector genes. Here, we review current understanding of TF-mediated gene regulation, discuss successful targeting strategies and highlight ongoing challenges and emerging approaches to address them.