👤 Spinel Karas

Inhibition of branched-chain ketoacid dehydrogenase kinase (BDK or BCKDK), a negative regulator of branched-chain amino acid (BCAA) metabolism, is hypothesized to treat cardio-metabolic diseases. From a starting point with potential idiosyncratic toxicity risk, modification to a benzothiophene core and discovery of a cryptic pocket allowed for improved potency with 3-aryl substitution to arrive at PF-07328948, which was largely devoid of protein covalent binding liability. This BDK inhibitor was shown also to be a BDK degrader in cells and in vivo rodent studies. Plasma biomarkers, including BCAAs and branched-chain ketoacids (BCKAs), were lowered in vivo with enhanced pharmacodynamic effect upon chronic dosing due to BDK degradation. This molecule improves metabolic and heart failure end points in rodent models. PF-07328948 is the first known selective BDK inhibitor candidate to be examined in clinical studies, with Phase 1 single ascending dose data showing good tolerability and a pharmacokinetic profile commensurate with once-daily dosing. Show less

5-Lipoxygenase (5-LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5-LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter-interacting proteins, obtained by DNA pulldown and label-free quantitative mass spectrometry. Protein preparations from myeloid and B-lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specific ALOX5 promotor binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel-like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G-quadruplex structures. As G-quadruplexes are implicated in gene regulation, spectroscopic and antibody-based methods were used to confirm their presence within the GC-rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form DNA G-quadruplex structures, which may play a functional role in ALOX5 gene regulation. Show less