Rational Design and Development of Selective BRD7 Bromodomain Inhibitors and Their Activity in Prostate Cancer.

Bromodomain-containing proteins are readers of acetylated lysine and play important roles in cancer. Bromodomain-containing protein 7 (BRD7) is implicated in multiple malignancies; however, there are no selective chemical probes to study its function in disease. Using crystal structures of BRD7 and BRD9 bromodomains (BDs) bound to BRD9-selective ligands, we identified a binding pocket exclusive to BRD7. We synthesized a series of ligands designed to occupy this binding region and identified two inhibitors with increased selectivity toward BRD7, 1-78 and 2-77, which bind with submicromolar affinity to the BRD7 BD. Our binding mode analyses indicate that these ligands occupy a uniquely accessible binding cleft in BRD7 and maintain key interactions with the asparagine and tyrosine residues critical for acetylated lysine binding. Finally, we validated the utility and selectivity of the compounds in cell-based models of prostate cancer.

Synthesis and evaluation of third generation vitamin D3 analogues as inhibitors of Hedgehog signaling.

The Hedgehog (Hh) pathway is a developmental pathway with therapeutic potential as a target for a variety of cancers. In recent years, several vitamin D-based compounds have been identified as potent inhibitors of Hh signaling. These analogues contain aromatic phenol A-ring mimics coupled to the CD-ring side chain of vitamin D3 through modified seco-B regions. To continue structure-activity relationship studies on this class of Hh pathway inhibitors, multiple series of vitamin D-based analogues that contain an amine-based seco-B tether and/or incorporate a hydroxyl moiety on C-25 were designed and synthesized. These compounds were evaluated in multiple cell lines for their anti-Hh activity, and we identify analogues 16, 21, 22 as potent vitamin D-based Hh inhibitors (IC50 values of 110-340 nM). We also performed a series of mechanism of action studies in knockout cell lines to further explore whether these analogues inhibit the Hh pathway through a known Hh pathway component or the vitamin D receptor. While the specific cellular target that mediates these effects remains elusive, our studies suggest multiple cellular targets may mediate the anti-Hh activity of this scaffold.

Small Molecule Targeting of Specific BAF (mSWI/SNF) Complexes for HIV Latency Reversal.

The persistence of a pool of latently HIV-1-infected cells despite combination anti-retroviral therapy treatment is the major roadblock for a cure. The BAF (mammalian SWI/SNF) chromatin remodeling complex is involved in establishing and maintaining viral latency, making it an attractive drug target for HIV-1 latency reversal. Here we report a high-throughput screen for inhibitors of BAF-mediated transcription in cells and the subsequent identification of a 12-membered macrolactam. This compound binds ARID1A-specific BAF complexes, prevents nucleosomal positioning, and relieves transcriptional repression of HIV-1. Through this mechanism, these compounds are able to reverse HIV-1 latency in an in vitro T cell line, an ex vivo primary cell model of HIV-1 latency, and in patient CD4+ T cells without toxicity or T cell activation. These macrolactams represent a class of latency reversal agents with unique mechanism of action, and can be combined with other latency reversal agents to improve reservoir targeting.

Synthesis and evaluation of vitamin D3 analogues with C-11 modifications as inhibitors of Hedgehog signaling.

Previous structure-activity relationship studies have provided potent and selective analogues of vitamin D3 as inhibitors of the Hedgehog (Hh) signaling pathway. These analogues contain both modified A- and seco-B ring motifs, and have been evaluated for anticancer therapeutic potential. To continue our studies on this scaffold, a new series of compounds were synthesized to explore additional interactions and spatial constraints. These compounds incorporate functional groups of varying size and hydrophobicity at the C-11 position. While large hydrophobic moieties (9c-e) resulted in significant loss of Hh inhibition, smaller or more flexible moieties (9a, 11) maintain anti-Hh activity. These results call for additional and continued studies to identify the binding pocket to better understand these structure-activity relationships.

Synthesis and Evaluation of Osteogenic Oxysterols as Hedgehog Pathway Activators.

Oxysterols (OHCs) are metabolic byproducts of cholesterol that are known to function as agonists of the Hedgehog (Hh) signaling pathway. Previously, we reported 23(S)-hydroxycholesterol [23(S)-OHC, 4] as a potent activator of Hh signaling with the ability to functionally differentiate mouse embryonic fibroblasts to an osteogenic fate. To obtain 23(S)-OHC in quantities suitable for in vivo evaluation, we developed a revised synthetic route that decreases the number of steps and chromatographic purifications, and which also enhances the stereoselective nature of the synthesis. This new route also allows access to the C21 methyl group of the OHC scaffold, and several new analogues with varying stereochemistry at this location were evaluated for their ability to up-regulate the Hh pathway.

Vitamin D3 analogues that contain modified A- and seco-B-rings as hedgehog pathway inhibitors.

The hedgehog (Hh) signaling pathway is a developmental signaling pathway that has been implicated as a target for anti-cancer drug development in a variety of human malignancies. Several natural and synthetic vitamin D-based seco-steroids have been identified as potent inhibitors of Hh signaling with chemotherapeutic potential. These include the previously characterized analogue 4, which contains the northern CD-ring/side chain region of vitamin D3 (VD3) linked to an aromatic A-ring mimic through an ester bond. To further explore structure-activity relationships for this class of VD3-based Hh pathway inhibitors, we have designed, synthesized and evaluated several series of compounds that modify the length, composition, and stereochemical orientation of the ester linker. These studies have identified compounds 54 and 55, which contain an amine linker and an aromatic A-ring incorporating a para-phenol, as new lead compounds with enhanced potency against the Hh pathway (IC50 values = 0.40 and 0.32 µM, respectively).