Mycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is a validated target for tuberculosis (TB) drug development. Here we report the X‐ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, which enabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities, and low risk of toxicity profile except for the inhibition of CYP2C9. A crystal structure of CYP2C9 in complex with a TCA1 analog revealed the similar interaction patterns to the DprE1–TCA1 complex. Guided by the structures, an optimized molecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of a clinical candidate to treat TB.

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.

Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell–recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021).

Mycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is av alidated target for tuberculosis (TB) drug development. Here we report the X-ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, whichenabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities,and lowrisk of toxicity profile except for the inhibition of CYP2C9. Ac rystal structure of CYP2C9 in complex with aT CA1 analog revealed the similar interaction patterns to the DprE1-TCA1 complex. Guided by the structures,a no ptimizedm olecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of ac linical candidate to treat TB., an essential cell wall component.[1] Conditional knockdown studies showed that loss of dprE1 results in as trong bactericidal effect in vitro.[2] Multiple covalent [3] and non-covalent [4] DprE1 inhibitors have been identified and showed in vitro and in vivo activities against Mtb,f urther validating DprE1 as an attractive anti-tuberculosis (TB) drug target. [5] We previously reported the identification of TCA1f rom ac ell-based phenotypic screen, and demonstrated it is aD prE1 inhibitor.[6] Since then we have generated new derivatives of TCA1 to improve its in vitro potency,P K properties,a nd in vivo efficacy. When analyzing toxicity profiles,wenoticed that many TCA1analogs show inhibition of CYP2C9, one of six major cytochrome P450 enzymes which determine the clearance of 75 %o fm arketed drugs.T herefore,i nhibition of P450 enzymes such as CYP2C9 can potentially lead to drug-drug interactions,w hich may cause serious issues for combinatory drug regimen for TB treatment. [7] Here we report the X-ray crystal structures of WT DprE1, aT CA1-resistant DprE1 mutant, and CYP2C9 in complex with TCA1 analogs.Along with structure activity relationship studies,t he mechanism of resistance by the mutant DprE1 enzyme and the tight correlation between DprE1 and CYP2C9 inhibition was elucidated. We further generated analogs with excellent in vitro and in vivo activities against Mtb,and selectively targeting DprE1 over CYP2C9.In the DprE1-TCA1 structure,T CA1s howed ap lanar conformation and tight fit in the DprE1 active site.T he benzothiazole core is oriented parallel to the FADi soalloxazine ring and the thiophene carboxamide resides in small hydrophobic pocket. Besides substantial hydrophobic interactions,m ultiple hydrogen bonding interactions are formed between TCA1a nd residues Lys418, His132, and Ser228 ( Figure S1 in the Supporting Information, SI). Guided by the structure,o ur medicinal chemistry effort focused on the benzothiazole core (red), thiophene carboxamide (green) and the acylcarbamate (blue) of TCA1 ( Figure 1).TheGln334 amide group and the Ty r60 hydroxy group ...