ABSTRACTor sink of atmospheric CO 2 depending on the rate of SOC formation and decomposition (Van Breemen andForested ecosystems have been identified as potential C sinks. Feijtel, 1990). It is therefore important to understand depleted (Johnson, 1992

Upregulation of the transcription factor Nrf2 by inhibition of the interaction with its negative regulator Keap1 constitutes an opportunity for the treatment of disease caused by oxidative stress. We report a structurally unique series of nanomolar Keap1 inhibitors obtained from a natural product-derived macrocyclic lead. Initial exploration of the structure-activity relationship of the lead, followed by structure-guided optimization, resulted in a 100-fold improvement in inhibitory potency. The macrocyclic core of the nanomolar inhibitors positions three pharmacophore units for productive interactions with key residues of Keap1, including R415, R483, and Y572. Ligand optimization resulted in the displacement of a coordinated water molecule from the Keap1 binding site and a significantly altered thermodynamic profile. In addition, minor reorganizations of R415 and R483 were accompanied by major differences in affinity between ligands. This study therefore indicates the importance of accounting both for the hydration and flexibility of the Keap1 binding site when designing high-affinity ligands.

Serine proteases comprise about one-third of all proteases, and defective regulation of serine proteases is involved in numerous diseases. Therefore, serine protease inhibitors are promising drug candidates. Aminomethyl diphenyl phosphonates have been regularly used as scaffolds for covalent serine protease inhibition and the design of activity-based probes. However, they cannot make use of a protease's primed site. Therefore, we developed a facile two-step synthesis toward a set of phenyl phosphinates, which is a related scaffold but can interact with the primed site. We tested their inhibitory activity on five different serine proteases and found that a phenyl group directly attached to the phosphorus atom leads to superior activity compared with phosphonates.

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Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC50 values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.

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