The reliance of many cancers on aerobic glycolysis has stimulated efforts to develop lactate dehydrogenase (LDH) inhibitors. However, despite significant efforts, LDH inhibitors (LDHi) with sufficient specificity and in vivo activity to determine whether LDH is a feasible drug target are lacking. We describe an LDHi with potent, on-target, in vivo activity. Using hyperpolarized magnetic resonance spectroscopic imaging (HP-MRSI), we demonstrate in vivo LDH inhibition in two glycolytic cancer models, MIA PaCa-2 and HT29, and we correlate depth and duration of LDH inhibition with direct anti-tumor activity. HP-MRSI also reveals a metabolic rewiring that occurs in vivo within 30 min of LDH inhibition, wherein pyruvate in a tumor is redirected toward mitochondrial metabolism. Using HP-MRSI, we show that inhibition of mitochondrial complex 1 rapidly redirects tumor pyruvate toward lactate. Inhibition of both mitochondrial complex 1 and LDH suppresses metabolic plasticity, causing metabolic quiescence in vitro and tumor growth inhibition in vivo.

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Toward this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First, we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tunable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.

Lysine acetyltransferases (KATs) are critical regulators of signaling in many diseases, including cancer. A major challenge in establishing the targetable functions of KATs in disease is a lack of well-characterized, cell-active KAT inhibitors. To confront this challenge, here we report a microfluidic mobility shift platform for the discovery and characterization of small molecule KAT inhibitors. Novel fluorescent peptide substrates were developed for four well-known KAT enzymes (p300, Crebbp, Morf, and Gcn5). Enzyme-catalyzed acetylation alters the electrophoretic mobility of these peptides in a microfluidic chip, allowing facile and direct monitoring of KAT activity. A pilot screen was used to demonstrate the utility of microfluidic mobility shift profiling to identify known and novel modulators of KAT activity. Real-time kinetic monitoring of KAT activity revealed that garcinol, a natural product KAT inhibitor used in cellular studies, exhibits time-dependent and detergent-sensitive inhibition, consistent with an aggregation-based mechanism. In contrast, the cell-permeable bisubstrate inhibitor Tat-CoA exhibited potent and time-independent KAT inhibition, highlighting its potential utility as a cellular inhibitor of KAT activity. These studies define microfluidic mobility shift profiling as a powerful platform for the discovery and characterization of small molecule inhibitors of KAT activity, and provide mechanistic insights potentially important for the application of KAT inhibitors in cellular contexts.