Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be ...particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets.
Myeloma is a plasma cell malignancy characterized by the overproduction of immunoglobulin, and is therefore susceptible to therapies targeting protein homeostasis. We hypothesized that heat shock ...factor 1 (HSF1) was an attractive therapeutic target for myeloma due to its direct regulation of transcriptional programs implicated in both protein homeostasis and the oncogenic phenotype. Here, we interrogate HSF1 as a therapeutic target in myeloma using bioinformatic, genetic, and pharmacologic means.
To assess the clinical relevance of HSF1, we analyzed publicly available patient myeloma gene expression datasets. Validation of this novel target was conducted in
experiments using shRNA or inhibitors of the HSF1 pathway in human myeloma cell lines and primary cells as well as in
human myeloma xenograft models.
Expression of HSF1 and its target genes were associated with poorer myeloma patient survival. ShRNA-mediated knockdown or pharmacologic inhibition of the HSF1 pathway with a novel chemical probe, CCT251236, or with KRIBB11, led to caspase-mediated cell death that was associated with an increase in EIF2α phosphorylation, CHOP expression and a decrease in overall protein synthesis. Importantly, both CCT251236 and KRIBB11 induced cytotoxicity in human myeloma cell lines and patient-derived primary myeloma cells with a therapeutic window over normal cells. Pharmacologic inhibition induced tumor growth inhibition and was well-tolerated in a human myeloma xenograft murine model with evidence of pharmacodynamic biomarker modulation.
Taken together, our studies demonstrate the dependence of myeloma cells on HSF1 for survival and support the clinical evaluation of pharmacologic inhibitors of the HSF1 pathway in myeloma.
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Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an ...efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.
CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription ...factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.
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ERAP1 is a zinc-dependent M1-aminopeptidase that trims lipophilic amino acids from the N-terminus of peptides. Owing to its importance in the processing of antigens and regulation of ...the adaptive immune response, dysregulation of the highly polymorphic ERAP1 has been implicated in autoimmune disease and cancer. To test this hypothesis and establish the role of ERAP1 in these disease areas, high affinity, cell permeable and selective chemical probes are essential. DG013A 1, is a phosphinic acid tripeptide mimetic inhibitor with reported low nanomolar affinity for ERAP1. However, this chemotype is a privileged structure for binding to various metal-dependent peptidases and contains a highly charged phosphinic acid moiety, so it was unclear whether it would display the high selectivity and passive permeability required for a chemical probe. Therefore, we designed a new stereoselective route to synthesize a library of DG013A 1 analogues to determine the suitability of this compound as a cellular chemical probe to validate ERAP1 as a drug discovery target.
Polypharmacology is often a key contributor to the efficacy of a drug, but is also a potential risk. We investigated two hits discovered via a cell-based phenotypic screen, the CDK9 inhibitor ...CCT250006 (1) and the pirin ligand CCT245232 (2), to establish methodology to elucidate their secondary protein targets. Using computational pocket-based analysis, we discovered intrafamily polypharmacology for our kinase inhibitor, despite little overall sequence identity. The interfamily polypharmacology of 2 with B-Raf was used to discover a novel pirin ligand from a very small but privileged compound library despite no apparent ligand or binding site similarity. Our data demonstrates that in areas of drug discovery where intrafamily polypharmacology is often an issue, ligand dissimilarity cannot necessarily be used to assume different off-target profiles and that understanding interfamily polypharmacology will be important in the future to reduce the risk of idiopathic toxicity and in the design of screening libraries.
High hit rates from initial ligand-observed NMR screening can make it challenging to prioritize which hits to follow up, especially in cases where there are no available crystal structures of these ...hits bound to the target proteins or other strategies to provide affinity ranking. Here, we report a reproducible, accurate, and versatile quantitative ligand-observed NMR assay, which can determine K d values of fragments in the affinity range of low μM to low mM using transverse relaxation rate R 2 as the observable parameter. In this study, we examined the theory and proposed a mathematical formulation to obtain K d values using non-linear regression analysis. We designed an assay format with automated sample preparation and simplified data analysis. Using tool compounds, we explored the assay reproducibility, accuracy, and detection limits. Finally, we used this assay to triage fragment hits, yielded from fragment screening against the CRBN/DDB1 complex.
A range of bicyclic gababutins were synthesised. Of these compounds (−)-(
11A) and (
20A) proved to have excellent levels of in vitro potency and were profiled in an in vivo model of neuropathic ...pain.
Synthesis of a number of bicyclic five-membered ring derivatives of gabapentin led to the identification of two compounds, (−)-(
11A) and (
20A) which both had an excellent level of potency against α
2δ and were profiled in an in vivo model of neuropathic pain.
High hit rates from initial ligand-observed NMR screening can make it challenging to prioritize which hits to follow up, especially in cases where there are no available crystal structures of these ...hits bound to the target proteins or other strategies to provide affinity ranking. Here, we report a reproducible, accurate, and versatile quantitative ligand-observed NMR assay, which can determine
values of fragments in the affinity range of low μM to low mM using transverse relaxation rate
as the observable parameter. In this study, we examined the theory and proposed a mathematical formulation to obtain
values using non-linear regression analysis. We designed an assay format with automated sample preparation and simplified data analysis. Using tool compounds, we explored the assay reproducibility, accuracy, and detection limits. Finally, we used this assay to triage fragment hits, yielded from fragment screening against the CRBN/DDB1 complex.
β-Sheet mediated protein-protein interactions are involved in key signalling pathways in diseases such as cancer. We present small molecule β-strand mimetics and investigate their interactions with a ...model tripeptide. Using (1)H NMR, the thermodynamic parameters for their binding are determined. These give insight into this biologically important interaction.
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