Mycobacterium Tuberculosis Methylcitrate Cycle Regulation and Study of Non-Covalent Natural Product Inhibitors of Proteasome

The methylcitrate cycle and glyoxylate bypass are two critical pathways of the central carbon metabolism of the human pathogen Mycobacterium tuberculosis (Mtb). Transcription of the key enzymes in these pathways is controlled by two paralogous local regulators, MtPrpR and MtRamB. No structure is available for these regulators or their homologs and the regulatory mechanisms are not well understood. In this study, we present the crystal structures of different forms of MtPrpR including two domains of unknown function. Our studies showed that MtPrpR is an iron-sulfur protein that binds to coenzyme A (CoA) and its derivatives. A single amino acid polymorphism in the CoA binding pocket enables the protein homologs to distinguish among different CoA derivatives. Our study suggested the methylcitrate cycle and glyoxylate bypass are regulated through a combination of feedforward and feedback mechanisms based on the CoA pool composition. The ubiquitin-proteasome pathway plays a pivotal role in cellular protein breakdown. Dysregulation of proteolysis leads to various human diseases including cancer. Inhibiting the proteasome is a promising approach in cancer treatment. Several inhibitors targeting the 20S proteasome have been tested on humans and approved by the FDA as antitumor drugs. In this study, we characterized a fungal derived natural product compound with a [M+H]⁺ m/z value of 721.33 (designated Mz721). Mz721 preferentially targets the trypsin- and chymotrypsin-like sites of the yeast 20S proteasome. The IC₅₀ value for the trypsin-like site in particular is below five nanomolar. The crystal structure of the yeast 20S proteasome in complex with Mz721 revealed that the inhibitor is structurally similar to the previously reported TMC-95 series natural products. It binds to all active sites in a non-covalent mode. Being cytotoxic against cancer cells, Mz721 is much less toxic than bortezomib, a first-in-class agent for multiple myeloma treatment, on the human dermal fibroblasts. As a result, Mz721 can act as a lead molecule to develop drugs that target the trypsin-like site of the 20S proteasome