Research projects

2009 Report:

TB Drug Discovery

We are leading a major effort to discover new drugs for the treatment of TB as part of the New Medicines for Tuberculosis Project, NM4TB, funded by the European Commission. In 2009, we described the identification and characterization of the 1,3-benzothiazin-4-ones (BTZ), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo and in murine models of TB. Using genetics and biochemistry, the enzyme decaprenylphosphoryl-b-D-ribose 2’-epimerase was identified as a major BTZ target. Inhibition of this enzymatic activity abolishes formation of decaprenylphosphoryl arabinose, a key precursor required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043 is a candidate for inclusion in combination therapies for both drug-sensitive and extensively-drug resistant TB.

Signal transduction, phosphorelays and cell wall biosynthesis in M. tuberculosis

There is mounting evidence that cell growth and elongation is controled by a phosphorelay involving serine-threonine protein kinases (STPK) and phosphatases, and forkhead-associated proteins that recognize phospho-threonine residues. We are intensively investigating the biological function of PknB, and trying to identify the ligand for this essential receptor kinase, a potential therapeutic target for new TB drugs. PknB localizes to the poles of tubercle bacilli.

Protein secretion and pathogenicity

The ESX-1 protein secretion system is the major virulence determinant operating in M. tuberculosis and has been lost by the vaccine strains M. bovis BCG and M. microti. ESX-1 is required for the export of small helical-hairpin proteins belonging to the ESAT-6 family as well as other effector proteins of unknown function. ESX-1 mediates host cell entry of tubercle bacilli and triggers intercell spread. We are using an integrated approach involving biochemistry, genetics, X-ray crystallography and electron microscopy to establish the organization, architecture, structure and function of this ATP-driven secretory apparatus. The figure shows crystals of key proteins from the ESX-1 system, which represents an attractive target for chemical biology and drug discovery.

explaination diagram
Figure. Crystals of key proteins from the ESX-1 system

A regulatory map of the M. tuberculosis genome

We have adopted an integrated approach to studying gene regulation by using chromatin-immunoprecipitation of DNA-binding proteins in conjunction with high density oligonucleotide-based microarrays or high-throughput sequencing to map the genome. Using this approach we demonstrated that the BlaI repressor controls expression of five separate genomic loci that respond to beta-lactam antibiotics and mapped all the RNA polymerase binding sites in the genome. Regulatory information is being incorporated into TubercuList, the genome server dedicated to M. tuberculosis http://tuberculist.epfl.ch/ , for which we are the official curators.

Phylogeography of leprosy

Despite the massive and highly successful implementation of mulitidrug therapy by the World Health Organisation, leprosy remains a serious public health problem in several countries probably due to our inability to identify infectious cases early enough. One of our goals is the development of an epidemiological tool to monitor transmission of the disease. This uses comparative genomics, particularly SNP (single nucleotide polymorphism) analysis of patient isolates, to monitor the phylogeography of leprosy. In collaboration with the WHO, we are also coordinating a worldwide effort to monitor the emergence of drug resistance.

 

2008 Report:

TB Drug Discovery

The Cole laboratory at EPFL is leading a major effort to discover new drugs for the treatment of tuberculosis (TB) as part of an INTEGRATED PROJECT funded by the European Commission for five years from January 2006. The New Medicines for Tuberculosis Project, NM4TB, follows the highly successful X-TB programme that generated over 60 publications and 30 crystal structures of important proteins and drug targets from Mycobacterium tuberculosis.

NM4TB aims to discover and develop new drugs for the treatment of TB through an integrated approach implemented by a team, that combines some of Europe’s leading academic TB researchers, with a major pharmaceutical company and three SMEs, all with a strong commitment to discovering new anti-infective agents. NM4TB has a comprehensive portfolio of potential and validated targets plus several novel, proprietary anti-TB agents in its pipeline. For more details consult the NM4TB website.

In collaboration with the Biomolecular Screening Platform, led by Dr. Gerardo Turcatti, we are conducting innovative whole cell and target-based screens of chemical libraries in order to identify lead compounds. These are characterized in terms of their IC50 for the target and MIC for M. tuberculosis; potential mutagenicity and cytotoxicity are then evaluated before conducting further pharmacological tests such as microsomal stability, cytochrome P450 inhibition and protein binding. Below we describe briefly some of the areas under study.

Signal transduction, phosphorelays and cell wall biosynthesis in M. tuberculosis

There is mounting evidence that cell growth and elongation is controled by a phosphorelay involving serine-threonine protein kinases (STPK) and phosphatases, and forkhead-associated proteins that recognize phospho-threonine residues. See figure. We are intensively investigating the biological function of PknB, and trying to identify the ligand for this essential receptor kinase, a potential therapeutic target for new TB drugs. Downstream signalling partners have been identified and their physiological roles are being uncovered. Several enzymes catalysing key steps in cell wall biogenesis are also being intensively investigated.

Protein secretion and pathogenicity

The ESX-1 protein secretion system is the major virulence determinant operating in M. tuberculosis and has been lost by the vaccine strains M. bovis BCG and M. microti. ESX-1 is required for the export of small helical-hairpin proteins belonging to the ESAT-6 family as well as other effector proteins of unknown function. ESX-1 mediates host cell entry of tubercle bacilli and triggers intercell spread. We are using an integrated approach to establish the organisation, architecture and function of this ATP-driven secretory apparatus and consider that, despite its non-essentiality, it represents an attractive target for chemical biology and drug discovery.

A regulatory map of the M. tuberculosis genome

We have adopted an integrated approach to studying gene regulation by using chromatin-immunoprecipitation of DNA-binding proteins in conjunction with high density oliogonucleotide-based microarrays to map the genome. This is shedding new light on various aspects of the physiology and behavior of tubercle bacilli and represents a useful adjunct to transcriptomics in drug discovery, particularly in providing clues to mechanism of action. Regulatory information is being incorporated into TubercuList, the genome server dedicated to M. tuberculosis http://tuberculist.epfl.ch, for which we are the official curators.

Phylogeography and immunodiagnosis of leprosy

Despite the massive and highly successful implementation of mulitidrug therapy by the World Health Organisation, leprosy remains a serious public health problem in several countries probably due to our inability to identify infectious cases early enough. One of our goals is the development of an epidemiological tool to monitor transmission of the disease. This uses comparative genomics, particularly SNP (single nucleotide polymorphism) andVNTR (variable number tandem repeat analysis of patient isolates, to monitor the phylogeography of leprosy. Immunodiagnostic approaches are also being pursued in an attempt to establish a test capable of distinguishing between exposure to Mycobacterium leprae and infection.

Funding

In addition to EPFL core funding, UPCOL gratefully acknowledges the financial support of the Fondation Raoul Follereau, the European Commission (LHSP-CT-2005-018923) and (HEALTH-F3-2007-201762), the Bill and Melinda Gates Foundation, and the National Institutes of Health (NIAID RO1-AI47197-01A1).

fhaB-pknB gene cluster

explaination diagram
Figure 1. Conserved structure of the fhaB-pknB gene cluster in actinobacteria. This cluster comprises five genes, except in Streptomyces coelicolor where pknA is missing: fhaA, forkhead-associated (FHA) protein A; fhaB, FHA protein B; pstP, phosphoserine/threonine protein phosphatase; rodA, cell division protein; pbpA, peptidoglycan biosynthesis protein; pknA, pknB, STPK. In all five bacteria the cluster is situated near oriC but transcribed convergently with respect to the direction of replication. Abbreviations:M. tb., M. tuberculosis; M. smeg., M. smegmatis; C. diphth., Corynebacterium diphtheriae; S. coelicol., Streptomyces coelicolor.