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.

Prostate cancer is the second most common cause of cancer related deaths in men in the United States. Pathogenesis is driven by the androgen receptor (AR), which has led to front-line treatment modalities that are based on androgen deprivation therapy (ADT). About 10-20% of all prostate cancers evolve to resist ADT and are classified as castration-resistant prostate cancer (CRPC) indicating the continued need for new treatment options. Bromodomain-containing protein 4 (BRD4) is an acetylated-chromatin associating protein that is involved in transcriptional elongation, mRNA splicing, epigenetic bookmarking, and super-enhancer activity. The BRD4 protein has been shown to both bind and colocalize with AR at androgen response elements (AREs) on chromatin. Furthermore, elevated BRD4 expression is prognostic of increased prostate specific antigen (PSA) levels following radical prostatectomy and is correlated with higher Gleason scores and poor overall survival. The intertwined activity of BRD4 with multiple essential driver mechanisms of prostate cancer suggests it may be a key target for developing novel therapeutics. Using our ultra-high throughput cell-based screening platform, which directly measures degradation of pathogenic proteins upon exposure to diverse chemical libraries, we identified a series of novel monovalent BRD4 degraders that was optimized to produce our lead compound PLX-3618. A cancer cell panel screen for antiproliferative effects of PLX-3618 indicated enhanced sensitivity in subsets of prostate cancer lines. PLX-3618 elicited selective, rapid, and deep degradation of BRD4 protein in prostate cancer cell models, without degrading the closely related BRD2 and BRD3 proteins. Addition of either proteosome or neddylation inhibitors blocked BRD4 degradation indicating a ubiquitin-proteosome system mediated clearance mechanism. Degradation of BRD4 led to sustained multimodal inhibition of the AR pathway and disruption of key oncogene enhancer networks. When compared to pan-BET inhibitors, treatment of select prostate cancer cell lines with PLX-3618 resulted in increased levels of the tumor suppressors p53 and p21, an aberrant DNA damage response, and substantially amplified apoptosis. Finally, in in vivo preclinical models of prostate cancer, PLX-3618 showed far superior efficacy over a pan-BET inhibitor. Taken together, the selective degradation of BRD4 via the potent monovalent degrader PLX-3618 represents a novel strategy in treating prostate cancer. Citation Format: Kenneth Steadman, Gregory S. Parker, Geoffray Leriche, Sarah Fish, Julia Toth, Mary E. Spalding, Elizabeth Daniele, Aleksandar Jamborcic, Xiaoming Li, E Adam Kallel, Farhana Barmare, Kenneth Chng, Erika Green, Michael Hocker, Elliot Imler, Yi Zhang, Peggy A. Thompson, Simon Bailey. PLX-3618, a potent, selective monovalent BRD4 degrader demonstrates activity in models of prostate cancer [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 421.

Abstract Targeted protein degradation using the endogenous Ubiquitin Proteasome System (UPS) represents a fundamentally new approach to drug discovery that potentially allows proteins that cannot be modulated by conventional small molecule inhibitors to be brought under therapeutic control. Plexium has developed the DELPhe platform, which combines solid phase synthesis of DNA encoded libraries with high-throughput ultra-miniaturized cell-based assays, to readily and cost-effectively identify both bifunctional (PROTAC) and monovalent degraders for traditionally undruggable targets including scaffolding proteins, protein-protein interactions and transcription factors. In addition to modulating previously “undruggable” proteins, degradation can lead to greater efficacy and a prolonged downstream signaling response, thereby addressing common obstacles seen with small molecule inhibitors. The bromodomain extra-terminal (BET) protein family are epigenetic readers that have been targeted using small molecule inhibitors. Compounds currently in development typically inhibit multiple BET family members. The bromodomain protein BRD4 is a transcriptional and epigenetic regulator that associates at super-enhancers, driving the expression of oncogenic proteins such as MYC that are critical for the pathogenesis of cancer. We describe here the use of Plexium's DELPhe platform to sample extensive chemical space and discover small molecule monovalent degraders that demonstrate selective and sustained degradation of BRD4. Near complete degradation was observed within 4 hours and DC50 potency of <10 nM was achieved for BRD4 without any appreciable degradation of the highly homologous BRD2 and BRD3 proteins. Degradation of BRD4 resulted in down regulation of MYC protein levels and potent anti-proliferative activity (20-200 nM) against a panel of tumor cell lines with activity superior to inhibition with the pan-BET inhibitor JQ1. Co-treatment with either proteosome or neddylation inhibitors blocked BRD4 degradation, suggesting that protein turnover is regulated by a Cullin-RING Ligase (CRL). Biochemical studies verified a direct interaction between the small molecule and BRD4, suggesting that binding promotes a conformational change that exposes key protein motifs to ubiquitination and degradation. Mutational analysis and a ubiquitin ligase-focused CRISPR screen were used to provide insights into defining the principles of degradation. Collectively, these data demonstrate that the Plexium DELPhe platform enables the discovery of selective and potent monovalent degraders of BRD4. Citation Format: Gregory S. Parker, Julia A. Toth, Geoffray Leriche, Simon Bailey, Kenneth Chng, Sara Fish, Aleks Jamborcic, Elizabeth Daniele, Erika Green, Michael Hocker, Adam Kallel, Peggy A. Thompson, Steven D. Brown, Kandaswamy Vijayan. Discovery of selective and potent BRD4 protein degraders using Plexium's DELPhe platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1283.

The unusual activity differences of carbon linked versus oxygen linked 2-substituted piperazines as α7 nicotinic acetylcholine receptor agonists led to a conformational study of several examples. The conformational preferences of which are absent from the literature. We report the first study and explanation of the conformational preference of 2-substiturted piperazines and show an example of how this preference controls binding in a pharmaceutically relevant case. In all cases the axial conformation for these 1-acyl and 1 aryl 2-substituted piperazines was found to be preferred. For the ether linked compounds, the axial conformation was found to be further stabilized by an intramolecular hydrogen bond. The axial orientation also places the basic and pyridyl nitrogens into a special orientation that closely mimics nicotine. Molecular modeling studies confirm that the R enantiomers of the compounds can bind to the α7 nicotinic acetylcholine receptor with the basic and pyridyl nitrogens colocalized with their counterparts in Epibatidine.

Background Chagas disease is a neglected tropical disease (NTD) caused by the eukaryotic parasite Trypanosoma cruzi. The current clinical and preclinical pipeline for T. cruzi is extremely sparse and lacks drug target diversity.Methodology/Principal Findings In the present study we developed a computational approach that utilized data from several public whole- cell, phenotypic high throughput screens that have been completed for T. cruzi by the Broad Institute, including a single screen of over 300,000 molecules in the search for chemical probes as part of the NIH Molecular Libraries program. We have also compiled and curated relevant biological and chemical compound screening data including (i) compounds and biological activity data from the literature, (ii) high throughput screening datasets, and (iii) predicted metabolites of T. cruzi metabolic pathways. This information was used to help us identify compounds and their potential targets. We have constructed a Pathway Genome Data Base for T. cruzi. In addition, we have developed Bayesian machine learning models that were used to virtually screen libraries of compounds. Ninety-seven compounds were selected for in vitro testing, and 11 of these were found to have EC50 < 10 mu M. We progressed five compounds to an in vivo mouse efficacy model of Chagas disease and validated that the machine learning model could identify in vitro active compounds not in the training set, as well as known positive controls. The antimalarial pyronaridine possessed 85.2% efficacy in the acute Chagas mouse model. We have also proposed potential targets (for future verification) for this compound based on structural similarity to known compounds with targets in T. cruzi.Conclusions/Significance We have demonstrated how combining chemoinformatics and bioinformatics for T. cruzi drug discovery can bring interesting in vivo active molecules to light that may have been overlooked. The approach we have taken is broadly applicable to other NTDs.

HSP90 continues to be a target of interest for neurodegeneration indications. Selective knockdown of the HSP90 cytosolic isoforms α and β is sufficient to reduce mutant huntingtin protein levels in vitro. Chemotype-dependent binding conformations of HSP90α/β appear to strongly influence isoform selectivity. The rational design of HSP90α/β inhibitors selective versus the mitochondrial (TRAP1) and endoplasmic reticulum (GRP94) isoforms offers a potential mitigating strategy for mechanism-based toxicities. Better tolerated HSP90 inhibitors would be attractive for targeting chronic neurodegenerative diseases such as Huntington’s disease.

Structure-activity relationship studies centered around 3'-substituted (Z)-5-(2'-(thienylmethylidene))1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-chromeno[3,4-f]quinolines are described. A series of highly potent and efficacious selective glucocorticoid receptor modulators were identified with in vitro activity comparable to dexamethasone. In vivo evaluation of these compounds utilizing a 28 day mouse tumor xenograft model demonstrated efficacy equal to dexamethasone in the reduction of tumor volume.

A series of selective androgen receptor modulators (SARMs) with a wide spectrum of receptor modulating activities was developed based on optimization of the 4-substituted 6-bisalkylamino-2-quinolinones (3). Significance of the trifluoromethyl group on the side chains and its interactions with amino acid residues within the androgen receptor (AR) ligand binding domain are discussed. A representative analog (9) was tested orally in a rodent model of hypogonadism and demonstrated desirable tissue selectivity.

A series of androgen receptor modulators based on 8H-[1,4]oxazino[2,3-f]quinolin-8-ones was synthesized and evaluated in an androgen receptor transcriptional activation assay. The most potent analogues from the series exhibited single-digit nanomolar potency in vitro. Compound 18h demonstrated full efficacy in the maintenance of muscle weight, at 10 mg/kg, with reduced activity in prostate weight in an in vivo model of androgen action.

A new structurally simple series of potent lipophilic aza-retinoids RXR agonists has been developed. SAR studies for the N-alkyl-azadienoic acids described here demonstrate that the RXR activity profile is sensitive to the N-alkyl chain length. Further, we have expanded the work to include azadienoic acids, which exhibited many accessible conformations leading to a better understanding of the SAR around the series.