Structural Characterization of Degrader-Induced Ternary Complexes Using Hydrogen-Deuterium Exchange Mass Spectrometry and Computational Modeling: Implications for Structure-Based Design.

The field of targeted protein degradation (TPD) has grown exponentially over the past decade with the goal of developing therapies that mark proteins for destruction leveraging the ubiquitin-proteasome system. One common approach to achieve TPD is to employ a heterobifunctional molecule, termed as a degrader, to recruit the protein target of interest to the E3 ligase machinery. The resultant generation of an intermediary ternary complex (target-degrader-ligase) is pivotal in the degradation process. Understanding the ternary complex geometry offers valuable insight into selectivity, catalytic efficiency, linker chemistry, and rational degrader design. In this study, we utilize hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify degrader-induced protein-protein interfaces. We then use these data in conjunction with constrained protein docking to build three-dimensional models of the ternary complex. The approach was used to characterize complex formation between the E3 ligase CRBN and the first bromodomain of BRD4, a prominent oncology target. We show marked differences in the ternary complexes formed in solution based on distinct patterns of deuterium uptake for two degraders, CFT-1297 and dBET6. CFT-1297, which exhibited positive cooperativity, altered the deuterium uptake profile revealing the degrader-induced protein-protein interface of the ternary complex. For CFT-1297, the ternary complexes generated by the highest scoring HDX-constrained docking models differ markedly from those observed in the published crystal structures. These results highlight the potential utility of HDX-MS to provide rapidly accessible structural insights into degrader-induced protein-protein interfaces in solution. They further suggest that degrader ternary complexes exhibit significant conformation flexibility and that biologically relevant complexes may well not exhibit the largest interaction surfaces between proteins. Taken together, the results indicate that methods capable of incorporating linker conformation uncertainty may prove an important component in degrader design moving forward. In addition, the development of scoring functions modified to handle interfaces with no evolved complementarity, for example, through consideration of high levels of water infiltration, may prove valuable. Furthermore, the use of crystal structures as validation tools for novel degrader methods needs to be considered with caution.

Evaluation of the Small-molecule BRD4 Degrader CFT-2718 in Small-cell Lung Cancer and Pancreatic Cancer Models.

Targeted, catalytic degradation of oncoproteins using heterobifunctional small molecules is an attractive modality, particularly for hematologic malignancies, which are often initiated by aberrant transcription factors and are challenging to drug with inhibitors. BRD4, a member of the bromodomain and extraterminal family, is a core transcriptional and epigenetic regulator that recruits the P-TEFb complex, which includes Cdk9 and cyclin T, to RNA polymerase II (pol II). Together, BRD4 and CDK9 phosphorylate serine 2 (pSer2) of heptad repeats in the C-terminal domain of RPB1, the large subunit of pol II, promote transcriptional elongation. Small-molecule degraders of BRD4 have shown encouraging efficacy in preclinical models for several tumor types but less efficacy in other cancers including small-cell lung cancer (SCLC) and pancreatic cancer. Here, we evaluated CFT-2718, a new BRD4-targeting degrader with enhanced catalytic activity and in vivo properties. In vivo, CFT-2718 has significantly greater efficacy than the CDK9 inhibitor dinaciclib in reducing growth of the LX-36 SCLC patient-derived xenograft (PDX) model and performed comparably to dinaciclib in limiting growth of the PNX-001 pancreatic PDX model. In vitro, CFT-2718 reduced cell viability in four SCLC and two pancreatic cancer models. In SCLC models, this activity significantly exceeded that of dinaciclib; furthermore, CFT-2718 selectively increased the expression of cleaved PARP, an indicator of apoptosis. CFT-2718 caused rapid BRD4 degradation and reduced levels of total and pSer2 RPB1 protein. These and other findings suggest that BRD-mediated transcriptional suppression merits further exploration in the setting of SCLC.

Identification of Highly Specific Diversity-Oriented Synthesis-Derived Inhibitors of Clostridium difficile.

In 2013, the Centers for Disease Control highlighted Clostridium difficile as an urgent threat for antibiotic-resistant infections, in part due to the emergence of highly virulent fluoroquinolone-resistant strains. Limited therapeutic options currently exist, many of which result in disease relapse. We sought to identify molecules specifically targeting C. difficile in high-throughput screens of our diversity-oriented synthesis compound collection. We identified two scaffolds with apparently novel mechanisms of action that selectively target C. difficile while having little to no activity against other intestinal anaerobes; preliminary evidence suggests that compounds from one of these scaffolds target the glutamate racemase. In vivo efficacy data suggest that both compound series may provide lead optimization candidates.

Assessment of Bromodomain Target Engagement by a Series of BI2536 Analogues with Miniaturized BET-BRET.

Evaluating the engagement of a small molecule ligand with a protein target in cells provides useful information for chemical probe optimization and pharmaceutical development. While several techniques exist that can be performed in a low-throughput manner, systematic evaluation of large compound libraries remains a challenge. In-cell engagement measurements are especially useful when evaluating compound classes suspected to target multiple cellular factors. In this study we used a bioluminescent resonant energy transfer assay to assess bromodomain engagement by a compound series containing bromodomain- and kinase-biasing polypharmacophores based on the known dual BRD4 bromodomain/PLK1 kinase inhibitor BI2536. With this assay, we discovered several novel agents with bromodomain-selective specificity profiles and cellular activity. Thus, this platform aids in distinguishing molecules whose cellular activity is difficult to assess due to polypharmacologic effects.

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells.

The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A. We observed increased intracellular concentrations of methylthioadenosine (MTA, the metabolite cleaved by MTAP) in cells harboring MTAP deletions. Furthermore, MTA specifically inhibited PRMT5 enzymatic activity. Administration of either MTA or a small-molecule PRMT5 inhibitor showed a modest preferential impairment of cell viability for MTAP-null cancer cell lines compared with isogenic MTAP-expressing counterparts. Together, our findings reveal PRMT5 as a potential vulnerability across multiple cancer lineages augmented by a common "passenger" genomic alteration.

Diversity-oriented synthesis probe targets Plasmodium falciparum cytochrome b ubiquinone reduction site and synergizes with oxidation site inhibitors.

BACKGROUND: The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways. METHODS: We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds. RESULTS: We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite. CONCLUSIONS: The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria.

Diversity-oriented synthesis-facilitated medicinal chemistry: toward the development of novel antimalarial agents.

Here, we describe medicinal chemistry that was accelerated by a diversity-oriented synthesis (DOS) pathway, and in vivo studies of our previously reported macrocyclic antimalarial agent that derived from the synthetic pathway. Structure-activity relationships that focused on both appendage and skeletal features yielded a nanomolar inhibitor of P. falciparum asexual blood-stage growth with improved solubility and microsomal stability and reduced hERG binding. The build/couple/pair (B/C/P) synthetic strategy, used in the preparation of the original screening library, facilitated medicinal chemistry optimization of the antimalarial lead.

Synthesis of stereochemically and skeletally diverse fused ring systems from functionalized C-glycosides.

A diversity-oriented synthesis (DOS) strategy was developed for the synthesis of stereochemically diverse fused-ring systems containing a pyran moiety. Each scaffold contains an amine and methyl ester for further diversification via amine capping and amide coupling. Scaffold diversity was evaluated in comparison to previously prepared scaffolds by a shape-based principal moments of inertia (PMI) analysis.

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria.

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure-activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.

Build/couple/pair strategy for the synthesis of stereochemically diverse macrolactams via head-to-tail cyclization.

A build/couple/pair (B/C/P) strategy was employed to generate a library of 7936 stereochemically diverse 12-membered macrolactams. All 8 stereoisomers of a common linear amine precursor were elaborated to form the corresponding 8 stereoisomers of two regioisomeric macrocyclic scaffolds via head-to-tail cyclization. Subsequently, these 16 scaffolds were further diversified via capping of two amine functionalities on SynPhase Lanterns. Reagents used for solid-phase diversification were selected using a sparse matrix design strategy with the aim of maximizing coverage of chemical space while adhering to a preset range of physicochemical properties.

STUDIES DIRECTED TOWARD THE SYNTHESIS OF NAKADOMARIN A.

The application of a Pummerer-initiated tandem reaction cascade leads to the highly stereoselective formation of the tetracyclic core of Nakadomarin A.

An unusual pathway to cyclobutane formation via desulfurative intramolecular photocycloaddition of an enone benzothiazoline pair.

Irradiation of the enone benzothiazoline 3 leads to the formation of cyclobutane 5. Preliminary mechanistic studies establish the intermediacy of an enecarbamate 14 in this photochemical transformation, which could be the result of sulfur extrusion from an episulfide intermediate. Photocycloaddition of the enecarbamate intermediate 14 leads to the formation of "crossed" photoadducts, i.e., 5, in excellent yield, with high levels of regio- and stereochemical control.