Pulmonary arterial hypertension (PAH) has demonstrated multi-serotonin receptor dependent pathologies, characterized by increased tone (5-HT1B receptor) and complex lesions (SERT, 5-HT1B, 5-HT2B receptors) of the pulmonary vasculature together with right ventricular hypertrophy, ischemia and fibrosis (5-HT2B receptor). Selective inhibitors of individual signaling elements – SERT, 5-HT2A, 5HT2B, and combined 5-HT2A/B receptors, have all been tested clinically and failed. Thus, inhibition of tryptophan hydroxylase 1 (TPH1), the rate limiting step in 5-HT synthesis, has been suggested as a more broad, and thereby more effective, mode of 5-HT inhibition. However, selectivity over non-pathogenic enzyme family members, TPH2, phenylalanine hydroxylase, and tyrosine hydroxylase has hampered therapeutic development. Here we describe the site/sequence, biochemical, and biophysical characterization of a novel allosteric site on TPH1 through which selectivity over TPH2 and related aromatic amino acid hydroxylases is achieved. We demonstrate the mechanism of action by which novel compounds selectively inhibit TPH1 using surface plasma resonance and enzyme competition assays with both tryptophan ligand and BH4 co-factor. We demonstrate 15-fold greater potency within a human carcinoid cell line versus the most potent known TPH1/2 non-specific inhibitor. Lastly, we detail a novel canine in vivo system utilized to determine effective biologic inhibition of newly synthesized 5-HT. These findings are the first to demonstrate TPH1-selective inhibition and may pave the way to a truly effective means to reduce pathologic 5-HT and thereby treat complex remodeling diseases such as PAH.

Sulfate‐reducing bacteria play a key role in the global carbon and sulfur cycles; a role realized through complex competitive or syntrophic metabolic relationships. These relationships are established in response to sulfate levels as well as concentrations of other nutrients such as short chain fatty acids (SCFAs). For some prokaryotes, SCFA production or consumption requires an ATP‐dependent reaction catalyzed by a SCFA kinase. In this study, we have determined the biochemical properties of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough heterologously expressed in E. coli. Our experiments characterize optimal conditions for activity as well as kinetic parameters for six SCFA substrates and the nucleotide cofactor ATP. These findings have implications on structure and function relationships as well as the potential physiological role of this enzyme. Support or Funding Information The authors would like to thank the Scott and Alice Thomson Fellowship Program for financial support of MB. Further, the authors thank the UW‐Parkside Committee on Research and Creative Activity and the College of Natural and Health Sciences for financial support of this project. Enzymatic Characterization of DvBuk. (A) DvBuk purity assessment was visualized by resolving 10 μg of purified DvBuk on 10% (w/v) polyacrylamide gel and stained with Coomassie Blue. Lane 1: Protein molecular weight markers, Lane 2: recombinant His‐tagged DvBuk. Enzyme activity (%) at various (B) pH and (C) temperature values. (D) Saturation kinetics of DvBuk with butyrate as substrate. (E) Relative activities of DvBuk in the presence of various SCFA substrates. Substrate Preference of DvBuk. (A) Relative activities of DvBuk in the presence of various SCFA substrates (relative activities normalized to observed specific activity in the presence of 200 mM valerate = 9.7 U mg−1). (B) Kinetic constants for DvBuk using various SCFA substrates.