Mechanisms of bacterial polyprenyl transferases and multi-target drug discovery for tuberculosis

Bacterial polyprenyl transferases transfer polyprenyl groups onto molecular acceptors, such as proteins and small molecules, and they are very important functional entities that are involved in bacterial cell wall biosynthesis, biofilm formation, virulence formation and natural product formation. Structurally, bacterial polyprenyl transferases can be categorized into eight groups: αcyclase, α, αβ, αβγ, βγ, e, ζ and TIM barrel fold proteins. Six bacterial polyprenyl transferases were investigated in the current research, including Streptomyces ghanaensis MoeO5 and Bacillus subtilis/Staphylococcus aureus PcrB (TIM barrel fold), Bacillus subtilis YisP (e fold), Bradyrhizobium japonicum Kaurene synthase (αcyclase fold), Mycobacterium tuberculosis Rv3378c and DPPS (ζ fold The function, mechanism of action, structure, inhibition and functional engineering of these six polyprenyl transferases were studied by X-ray crystallography, mutagenesis, activity assays, thermal dynamics measurements etc. Besides that, a multi-target drug discovery approach was also proposed as an attempt to combat drug resistance in anti-infective drug discovery campaigns, both based on structural homology of Mycobacterium tuberculosis Rv3378c and DPPS (ζ fold), and the multi-target effect of SQ-109 and its analogs. MoeO5 catalyzes the first step biosynthesis of Moenomycin A, which is a phosphoglycolipid antibiotic. We determined the first crystal structure of MoeO5 and studied the binding mode of bound substrates. We also assayed its activity with different substrates and compared MoeO5 with its homolog PcrB, which led to a proposal of MoeO5 reaction mechanism. YisP is essential in biofilm formation in Bacillus subtilis and is predicted to produce C30 isoprenoids. We determined the structure of YisP, and proved that YisP acts as a phosphatase. Using DSC, we confirmed that farnesol shift and broaden the gel-to-liquid crystal transition of lipids. The X-ray structure of the bacterial diterpene cyclase ent-kaur-16-ene synthase from the soil bacterium Bradyrhizobium japonicum was determined in apo-, substrate and inhibitor-bound forms. The catalytic activity of the cyclase was studied by site-directed mutagenesis and inhibition of the enzyme was also investigated. Structures of tuberculosinol/iso-tuberculosinol synthase (Rv3378c) and cis-decaprenyl diphosphate synthase (DPPS) from M. tuberculosis were determined. They are targets for anti-infective therapies that block virulence factor formation and for cell wall biosynthesis, respectively. Given the similarity in local and global structure between these two proteins, the possibility exists that it may be possible to develop inhibitors that target not only virulence, but also cell wall biosynthesis. SQ-109, one new anti-tuberculosis ethylenediamine drug, was found to be active against other bacteria and yeasts. Based on the SAR study and biochemistry assay results with eleven SQ-109 analogs, we found that it might be an example of multi-target drug candidate that inhibits isoprenoid/quinone…