Universitą degli studi di Pavia

 

Contenuto della pagina

 

De Rossi research activity

New antitubercular drugs: study of the cellular target and mechanism of action and resistance

Mycobacteria cause several diseases, including tuberculosis (TB), leprosy, and systemic infectionsin AIDS patients. The treatment of mycobacterial infectionsis often difficult because these bacteria are intrinsically resistantto most common antibiotics. TB has become an increasing health problem since the emergence of HIV, the increasing appearance of multidrug resistant (MDR) Mycobacterium tuberculosis strains, and the increased incidence of M. avium complex infections in HIV-infected individuals. Drug resistance has been observed for all five of the first-line anti-tuberculosis drugs isoniazid, rifampin, pyrazinamide, streptomycin, and ethambutol, and for several of the second-line anti-tuberculosis drugs. In addition, a grave public health threat, especially in populations with high rates of HIV and where there are few health care resources, is posed by extensively drug resistant (XDR) strains. These strains are MDR also resistant to any fluoroquinolone and to at least one of three injectable second-line drugs used in TB treatment (capreomycin, kanamicin, and amikacin) and leave patients (including many people living with HIV) virtually untreatable. Therefore, new drugs against new targets are urgently needed.

Two research lines are in progress:
 

1) Identification and characterization of the cellular target of pyrrole derivatives
BM212, belonging to a series of pyrrole derivatives, shows a potent activity against M. tuberculosis, non-tuberculosis mycobacteria, such as M. avium, and drug-resistant clinical isolates of M. tuberculosisas well as on intracellular bacilli. On the basis of these results, BM212 was considered the lead compound of this new class of derivatives and one of the promising future antimycobacterial drug. Since the target of BM212 was unknown, the objective of this work is the identification and characterization of the cellular target of the compound in M. smegmatis. To this aim, M. smegmatismutants resistant to BM212 have been isolated.To identify the gene(s) responsible for the resistance, a genomic library of one of these mutants has been constructedand screened for BM212 resistance.Two point mutations, giving rise to amino acid substitutions, have been identified in the mmpL3gene coding for the membrane protein MmpL3 with unknown function. Since mutations in the target gene are responsible for drug resistance, the MmpL3 protein seems to be the cellular target of BM212.The members of the MmpL family belong to the RND family of efflux pumps, membrane proteins of Gram negative bacteria, able to extrude out of the cell structurally and functionally unrelated compounds. Microbiological assays and uptake/efflux experiments demonstrated that MmpL3 does not extrude BM212 out of the cellmeaning that theprotein is not an efflux pump but the cellular target of BM212.Given the identification of mutations in mmpL3as responsible for resistance to BM212, the sequence of the mmpL3gene from BM212-resistant M. bovisBCG and M. tuberculosisisolates has been determined. All revealed substitutions non-synonymus mmpL3mutations.
By further characterization of the MmpL3 protein, it will be possible to obtain more insights to design new and more effective antimycobacterial BM212 derivatives.
 

2) Identification and characterization of the cellular target of new antitubercular molecules
This research is part of the project "New Medicines for Tuberculosis – NM4TB" funded by EC-VI FP. The cellular target of a new drug, belonging to the class of benzothiazinones (BTZ-NO2) has been identified in the M. tuberculosisRv3790 enzyme.. Both the over-expression and the mutation of a cysteine residue at the C-terminal of Rv3790 are responsible for high resistance levels to BTZ-NO2. It has been previously demonstrated that Rv3790 works in concert with Rv3791 in the biosynthesis of arabinogalactan, an important component of mycobacterial cell wall. In particular they are involved in the formation of decaprenyl-P-arabinose (DPA), a fundamental precursor in the arabinan synthesis. By enzymatic assay it was confirmed that the synthesis of DPA is inhibited in the presence of BTZ-NO2in wild type mycobacterial strains but not in BTZ-NO2resistant mutants.
The M. smegmatisNfnB nitroreductase, able to convert the original effective molecule BTZ-NO2into its less active derivative BTZ-NH2has been also identified. By further characterization of the Rv3790 and NfnB proteins, it will be possible to obtain more insights to designnew and more effective benzothiazinones derivatives.
 
A new project funded by EC-VII FP (More Medicines for Tuberculosis – MM4TB) started in February 2011 with the aim to identify new targets for new antitubercular drug
 
 
Operazione Trasparenza Realizzato con il CMS Ariadne Content Manager da Ariadne

Torna all'inizio