Full-Length NAD+-I Riboswitches Bind a Single Cofactor but Cannot Discriminate against Adenosine Triphosphate.

Bacterial riboswitches are structured RNAs that bind small metabolites to control downstream gene expression. Two riboswitch classes have been reported to sense nicotinamide adenine dinucleotide (NAD+), which plays a key redox role in cellular metabolism. The NAD+-I (class I) riboswitch stands out because it comprises two homologous, tandemly arranged domains. However, previous studies examined the isolated domains rather than the full-length riboswitch. Crystallography and ligand binding analyses led to the hypothesis that each domain senses NAD+ but with disparate equilibrium binding constants (KD) of 127 µM (domain I) and 3.4 mM (domain II). Here, we analyzed individual domains and the full-length riboswitch by isothermal titration calorimetry to quantify the cofactor affinity and specificity. Domain I senses NAD+ with a KD of 24.6 ± 8.4 µM but with a reduced ligand-to-receptor stoichiometry, consistent with nonproductive domain self-association observed by gel-filtration chromatography; domain II revealed no detectable binding. By contrast, the full-length riboswitch binds a single NAD+ with a KD of 31.5 ± 1.5 µM; dinucleotides NADH and AP2-ribavirin also bind with one-to-one stoichiometry. Unexpectedly, the full-length riboswitch also binds a single ATP equivalent (KD = 11.0 ± 3.5 µM). The affinity trend of the full-length riboswitch is ADP = ATP > NAD+ = AP2-ribavirin > NADH. Although our results support riboswitch sensing of a single NAD+ at concentrations significantly below the intracellular levels of this cofactor, our findings do not support the level of specificity expected for a riboswitch that exclusively senses NAD+. Gene regulatory implications and future challenges are discussed.

Selective and Cell-Active PBRM1 Bromodomain Inhibitors Discovered through NMR Fragment Screening.

PBRM1 is a subunit of the PBAF chromatin remodeling complex that uniquely contains six bromodomains. PBRM1 can operate as a tumor suppressor or tumor promoter. PBRM1 is a tumor promoter in prostate cancer, contributing to migratory and immunosuppressive phenotypes. Selective chemical probes targeting PBRM1 bromodomains are desired to elucidate the association between aberrant PBRM1 chromatin binding and cancer pathogenesis and the contributions of PBRM1 to immunotherapy. Previous PBRM1 inhibitors unselectively bind SMARCA2 and SMARCA4 bromodomains with nanomolar potency. We used our protein-detected NMR screening pipeline to screen 1968 fragments against the second PBRM1 bromodomain, identifying 17 hits with Kd values from 45 µM to >2 mM. Structure-activity relationship studies on the tightest-binding hit resulted in nanomolar inhibitors with selectivity for PBRM1 over SMARCA2 and SMARCA4. These chemical probes inhibit the association of full-length PBRM1 to acetylated histone peptides and selectively inhibit growth of a PBRM1-dependent prostate cancer cell line.