Development of Inhibitors of SAICAR Synthetase (PurC) from Mycobacterium abscessus Using a Fragment-Based Approach.

Mycobacterium abscessus (Mab) has emerged as a challenging threat to individuals with cystic fibrosis. Infections caused by this pathogen are often impossible to treat due to the intrinsic antibiotic resistance leading to lung malfunction and eventually death. Therefore, there is an urgent need to develop new drugs against novel targets in Mab to overcome drug resistance and subsequent treatment failure. In this study, SAICAR synthetase (PurC) from Mab was identified as a promising target for novel antibiotics. An in-house fragment library screen and a high-throughput X-ray crystallographic screen of diverse fragment libraries were explored to provide crucial starting points for fragment elaboration. A series of compounds developed from fragment growing and merging strategies, guided by crystallographic information and careful hit-to-lead optimization, have achieved potent nanomolar binding affinity against the enzyme. Some compounds also show a promising inhibitory effect against Mab and Mtb. This work utilizes a fragment-based design and demonstrates for the first time the potential to develop inhibitors against PurC from Mab.

Cis Selective RCM Study to the 14-Membered Cyclic Subunit of Bielschowskysin.

A concise, (Z)-selective ring-closing metathesis (RCM) route to the 14-membered carbocycle of bielschowskysin is detailed using naturally occurring chiral starting materials. Unproductive RCM substrates were attributed to alkyne chelation of the ruthenium catalyst and steric disadvantages within the cembranoid precursors, which was eventually circumvented by using cyclic diol benzylidene protection involving a C8-quaternary carbinol center.

Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO-E2-E3 Ligase Ternary Complexes.

E2 conjugating enzymes are the key catalytic actors in the transfer of ubiquitin, SUMO, and other ubiquitin-like modifiers to their substrate proteins. Their high rates of transfer and promiscuous binding complicate studies of their interactions and binding partners. To access specific, covalently linked conjugates of the SUMO E2 conjugating enzyme Ubc9, we prepared synthetic variants bearing site-specific non-native modifications including the following: (1) replacement of Cys93 to 2,3-diaminopropionic acid to form the amide-linked stable E2-SUMO conjugate, which is known to have high affinity for E3 ligases; (2) a photoreactive group (diazirine) to trap E3 ligases upon UV irradiation; and (3) an N-terminal biotin for purification and detection. To construct these Ubc9 variants in a flexible, convergent manner, we combined the three leading methods: native chemical ligation (NCL), α-ketoacid-hydroxylamine (KAHA) ligation, and serine/threonine ligation (STL). Using the synthetic proteins, we demonstrated the selective formation of Ubc9-SUMO conjugates and the trapping of an E3 ligase (RanBP2) to form the stable, covalently linked SUMO1-Ubc9-RanBP2 ternary complex. The powerful combination of ligation methods-which minimizes challenges of functional group manipulations-will enable chemical probes based on E2 conjugating enzymes to trap E3 ligases and facilitate the synthesis of other protein classes.