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SELECTION OF NEW TARGET PROTEINS
FOR DRUG DESIGN IN GENOME OF
MYCOBACTERIUM TUBERCULOSIS
Alexander V. Veselovsky
V.N. Orechovich Institute of Biomedical Chemistry RAMS, Moscow, Russia
e-mail: [email protected]
Modern pipeline of new drug development
Identify disease
Find a drug effective
against disease protein
(2-5 years)
Isolate protein
involved in
disease (2-5 years)
Preclinical testing
(1-3 years)
Human clinical trials
(2-10 years)
Formulation &
Scale-up
Ability to decreasing finance and time cost
+
-
FDA approval
(2-3 years)
Pipeline of target-based and main steps in drug development
Genomics for drug discovery
Genome
Annotation and
classification of genes
Drug targets
selection
Comparative genomics
Human genome
Gram(+) bacteria
genome
Genes-targets of
bacteria that differ
from human genes
D.T.Moir et al., 1999
Gram(-) bacteria
genome
Requirements of “Ideal” Antimicrobial Agent and to Its Target
Target selection (Comparative genomics)
favourable similarity
Unfavourable similarity
Human
genome
Genomes of
related species
Target
genome
Genomes of
other strains of
target species
Proteins with known
spatial structures
(PDB)
Genomes of human
symbiont
microorganisms
GeneMesh – program for protein-targets selection for antimicrobial drug discovery
using comparative and functional genomics
A.V. Dubanov, A.S. Ivanov, A.I. Archakov (2001) Computer searching of new targets for antimicrobial drugs
based on comparative analysis of genomes. Vopr. Med. Khim. 47, 353-367. (in Russian).
Algorithm of program GenMesh
GenMesh
Set of proteins
from PDB
BLAST
Spatial structure
ability
Genomes of
related species
Target genome
BLAST
BLAST
databases
Genomes of
other strains of
target species
Human genome
BLAST
BLAST
Presence of homologs
in genomes of
related species
Absence of mutations
in other strains of
target species
Absence of homologs
in human genome
Target selection in Mycobacterium tuberculosis H37Rv using broadened
set of genomes for analysis
targets for antimycobacterial agents without
influencing normal human microflora
Common targets for Mycobacteria and fungi
3D protein structure modelling
Approach
Homology
modelling
Limitation
Model and template sequence
identity must be > 30%
Results heavily dependent on
Threading
human expertise and information
(Fold recognition) from other methods for elimination
decoy folds
Ab initio
(De novo)
< 150 amino acids
* - RMSD of C (A) and residues true positions (%)
Accuracy*
1-3 A
80-95%
3-6 A
30-50%
4-8 A
< 30%
Target selection in genome of Mycobacterium tuberculosis H37Rv
Potential Targets Found in Genome of M. tuberculosis H37R
Freiberg C, Wieland B, Spaltmann F, Ehlert K, Brötz H, Labischinski H.Identification of novel essential Escherichia coli genes
conserved among pathogenic bacteria. J Mol Microbiol Biotechnol. 2001 Jul;3(3):483-9.
Thanassi JA, Hartman-Neumann SL, Dougherty TJ, Dougherty BA, Pucci MJ. Identification of 113 conserved essential genes using
a high-throughput gene disruption system in Streptococcus pneumoniae. Nucleic Acids Res. 2002 Jul 15;30(14):3152-62.
Russian Federal
Space Agency
Program for protein crystallization
in weightlessness
International space station (ISC)
Target M. tuberculosis H37R
Phosphopantetheine adenylyltransferase of bacteria
PPAT
4'-phosphopantetetheine + ATP
PPi + 3'dephospho-CoA
+ Pi
Coenzyme A
Penultimate and rate-limited enzyme of bacterial
coenzyme A biosynthesis
Comparison of spatial structures of PPAT M.tuberculosis
Active site
Green – from Russia (1,6 A)
Yellow – 1TFU.pdb (1,99 A)
Scheme of virtual screening for new PPAT inhibitors
in molecular database
Molecular database
Experimental testing
Database preprocessing
Manual selection
Docking
Compounds selection by scoring
functions consensus
Calculation of
additional scoring
function
Discovery ligands from molecular database by docking method
Empirical scoring function
The method is
fast
semi-automated
is applicable to 3-D models
does not need extensive training
Accuracy of scoring function
Relationship between scoing functions
Limitation of scoring functions
Srt
Ligand in
solution
HLW
Receptor
HRW
Free energy
bound water
free rotation
Sint
G = H-TS
loosely associated
water molecules
free water
Entropy
HLR
 SW
Svib
Receptor-Ligand complex
Enthalpy
Consensus of scoring functions
The first docking of compounds in PPAT active site
17500 complexes
Active site of phosphopantetheine adenylyltransferase M.tuberculosis
The second docking of compounds in PPAT active site
24000 complexes
Experimental testing of selected ligands
Acknowledgments. This work was supported in part by
Russian Federal Space Agency (in frame of ground
preparation of space research).
Participants:
Institute of Bioorganic Chemistry RAS
Institute of Crystallography RAS
Institute of Biomedical Chemistry RAMS