Mapping protein binding sites by photoreactive fragment pharmacophores.

Fragment screening is a popular strategy of generating viable chemical starting points especially for challenging targets. Although fragments provide a better coverage of chemical space and they have typically higher chance of binding, their weak affinity necessitates highly sensitive biophysical assays. Here, we introduce a screening concept that combines evolutionary optimized fragment pharmacophores with the use of a photoaffinity handle that enables high hit rates by LC-MS-based detection. The sensitivity of our screening protocol was further improved by a target-conjugated photocatalyst. We have designed, synthesized, and screened 100 diazirine-tagged fragments against three benchmark and three therapeutically relevant protein targets of different tractability. Our therapeutic targets included a conventional enzyme, the first bromodomain of BRD4, a protein-protein interaction represented by the oncogenic KRasG12D protein, and the yet unliganded N-terminal domain of the STAT5B transcription factor. We have discovered several fragment hits against all three targets and identified their binding sites via enzymatic digestion, structural studies and modeling. Our results revealed that this protocol outperforms screening traditional fully functionalized and photoaffinity fragments in better exploration of the available binding sites and higher hit rates observed for even difficult targets.

Inhibitor Library Screening of SH2 Domains Through Denaturation-Based Assays.

Screening of inhibitor libraries for candidate ligands is an important step in the drug discovery process. Thermal denaturation-based screening strategies are built on the premise that a protein-ligand complex has an altered stability profile compared to the protein alone. As such, these assays provide an accessible and rapid methodology for stratifying ligands that directly engage with the protein target of interest. Here, we describe three denaturation-based strategies for examining protein-inhibitor binding, in the context of SH2 domains. This includes conventional dye-based Thermal Shift Assays (TSA), nonconventional labeled ligand-based TSA, and Cellular Thermal Shift Assays (CETSA). Conventional dye-based TSA reports on the fluorescence of an external hydrophobic dye as it interacts with heat-exposed nonpolar protein surfaces as the temperature is incrementally increased. By contrast, nonconventional-labeled ligand TSA involves a fluorescence-tagged probe (phosphopeptide for SH2 domains) that is quenched as it dissociates from the protein during the denaturation process. CETSA involves monitoring the presence of the protein via Western blotting as the temperature is increased. In all three approaches, performing the assay in the presence of a candidate ligand can alter the melting profile of the protein. These assays offer primary screening tools to examine SH2 domain inhibitors libraries with varying chemical motifs, and a subset of the advantages and limitations of each approach is also discussed.

Fluorescence Polarization-Based Competition Assays to Evaluate Histone Deacetylase 6 Inhibitors.

Histone deacetylase 6 (HDAC6) is an emerging clinical target for the treatment of several hematological cancers and central nervous system disorders. HDAC6 catalyzes the deacetylation of lysine residues on substrates such as tubulin, with profound implications in key cellular processes, including cellular motility and migration. This critical deacetylation activity occurs at the catalytic domain 2 (CD2) of HDAC6, and small molecule inhibitors of HDAC6 are designed to target CD2. We briefly highlight previously reported strategies for recombinant bacterial expression and purification of the HDAC6 CD2. We aim to discuss competition assays that have been used to evaluate the potency of potential HDAC6 inhibitors against CD2 via displacement of pre-bound fluorescent HDAC-probes. Moreover, we elaborate on previous protocols that have been employed in inhibitor screening and present an HDAC6-selective probe that also enables rapid and reliable high-throughput screening of new chemical entities designed to target the HDAC6 CD2.