DRUG DEVELOPMENT AND OUT LINE OF DRUG

INVENTION AGAINST LEISHMANIA DONOVANI

Biomadical infomatics center Rajendra Memorial Research Institute of Medical

Science (RMRIMS)

Moonmoon DebPh.D scholarNIT Rourkela

Life Science Department509LS102

INDEX Drug discovery Protein homology modeling Leishmania sp. and Kala-azar Out line of drug development against

Leishmania donovani DNA isolation from Leishmania donovani

BIOINFORMATICS AND DRUG DISCOVERY

Drug discovery take years to decade for discovering a new drug and very costly

Effort to cut down the research timeline and cost by reducing wet-lab experiment use computer modeling.

MODERN DRUG DISCOVERY

small suitable molecule↓

manipulate structure to increase potency

↓*optimization of lead molecule into candidate drug*

fulfillment of required pharmacological properties:potency, absorption, bioavailability, metabolism, safety

↓clinical trials

THE DRUG DISCOVERY PROCESS(MODERN METHODS OF DRUG DISCOVERY)

Drug Target Identification Target Validation Lead Compound Identification Lead Optimization Preclinical and Clinical

Development

DRUG TARGET IDENTIFICATION

The identification of new, clinically relevant, molecular targets is a key step to the discovery of innovative drugs

many more drug targets exist! How to identify them?

Besides classical methods of cellular and molecular biology, new techniques of target identification are becoming increasingly important. These include:a) genomics b) bioinformaticsc) proteomics

a) genomics evolved from 2 independent advances:

1) Automation – resulting in a significant increase in the number of experiments that could be constructed in a given time. (eg. DNA sequencing)

2) Informatics- the ability to transform raw data into meaningful information by applying computerized techniques for managing, analyzing, and interpreting data

the identification of new biological targets has benefited from the genomics approach: eg. The sequencing of the human genome

blueprint of all proteins bioinformatics methods are used to transform the raw sequence into

meaningful information (eg. genes and their encoded proteins) and to compare with the whole genomes

b) Bioinformatics – the in silico identification of novel drug targets is now feasible by systematically searching for paralogs (related proteins within an organism) of known drug targets (eg. may be able to modify an existing drug to bind to the paralog).

Can compare the entire genome of pathogenic and nonpathogenic strains of a microbe and identify genes/proteins associated with pathogenism.

c) Proteomics – concerning expression analysis, it has been shown that the correlation between RNA and protein expression

It is becoming increasingly evident that the complexity of biological systems lies at the level of the proteins, and that genomics alone will not suffice to understand these systems.

It is also at the protein level that disease processes become manifest, and at which most (91%) drugs act.

Therefore, the analysis of proteins (including protein-protein, protein-nucleic acid, and protein-ligand interactions) will be utmost importance to target discovery.

Target identification with proteomics is performed by comparing the protein expression levels in normal and diseased tissues.

2) TARGET VALIDATION Involves demonstrating the relevance of

the target protein in a disease process / pathogenicity and ideally requires both gain and loss of function studies.

This is accomplished primarily with knock-out or knock-in animal models, small molecule inhibitors, antisense nucleic acid constructs, and neutralizing antibodies

3) LEAD COMPOUND IDENTIFICATION Compounds are mainly identified

using random (screening) or rational (design) approaches.

TRADITIONAL METHODS OF DRUG DISCOVERY

natural (plant-derived) treatment for illness/ailments

↓isolation of active compound

(small, organic)

synthesisof compound

↓

manipulation of structure to get better drug(greater efficacy, fewer side effects)

STRUCTURE BASED DRUG DESIGN Three dimensional structures of compounds from virtual

or physically existing libraries are docked into binding sites of target proteins with known or predicted structure.

Scoring functions evaluate the steric and electrostatic complementarity between compounds and the target protein.

The highest ranked compounds are then suggested for biological testing.

drug targets (usually proteins) binding of ligands to the target (docking)

↓

“rational” drug design

(benefits = saved time and money )

Ligand database Target Protein

Molecular docking

Ligand docked into protein’s active site

4) LEAD OPTIMIZATION

Only if the hits fulfill certain criteria are they regarded as leads. The criteria can originate from:

1) Pharmacodynamic properties - efficacy, potency, selectivity

2) Physiochemical properties - water solubility, chemical stability

3) Pharmacokinetic properties - metabolic stability and toxological aspects.

4) Chemical optimization potential - ease of chemical synthesis and derivatization.

5) Clinical Trials:Phase I: Single Ascending Dose (SAD) studiesMultiple Ascending Dose (MAD) studiesFood effect

80% of drugs fail the Phase I clinical trial.Phase IIPhase III

HOMOLOGY MODELING DEFINED Homology modeling

Based on the reasonable assumption that two homologous proteins will share very similar structures.

Given the amino acid sequence of an unknown structure and the solved structure of a homologous protein, each amino acid in the solved structure is matched computationally, into the corresponding amino acid from the unknown structure.

Homology Modeling Limitations Cannot study conformational changes Cannot find new catalytic/binding sites

WHY HOMOLOGY MODELING?

Value in structure based drug design Find common catalytic sites/molecular

recognition sites Use as a guide to planning and

interpreting experiments 70-80 % chance a protein has a similar

fold to the target protein due to X-ray crystallography or NMR spectroscopy

Sometimes it’s the only option or best guess

STEPS OF HOMOLOGY MODELING

Target protein search Click on structure Display fasta formet Save on note pad Run blast Protein data bank Blast % of identity ExPasy tool http://au.expasy.org/tools/ExPASy Proteomics Server

Tertiary structure prediction Homology modelingSWISS-MODEL  - An automated knowledge-based protein modelling server3Djigsaw - Three-dimensional models for proteins based on homologues of known structureCPHmodels - Automated neural-network based protein modelling serverESyPred3D - Automated homology modeling program using neural networksGeno3d - Automatic modelling of protein three-dimensional structureSDSC1 - Protein Structure Homology Modeling ServerThreadingPhyre (Successor of 3D-PSSM) - Automated 3D model building using profile-profile matching and secondary structureFugue - Sequence-structure homology recognitionHHpred - Protein homology detection and structure prediction by HMM-HMM comparisonLibellula - Neural network approach to evaluate fold recognition resultsLOOPP - Sequence to sequence, sequence to structure, and structure to structure alignmentSAM-T02 - HMM-based Protein Structure PredictionThreader - Protein fold recognitionSWEET - Constructing 3D models of saccharides from their sequencesAb initioHMMSTR/Rosetta - Prediction of protein structure from sequence

Then click on SWISS-MODEL Then click on automated model Email ID Project title and

copy pest the sequence

Discovery studio

LEISHMANIA SP. AND KALA-AZAR

Leishmania sp. is responsible for KALA-AZAR (KA) or Leishmaniasis Leishmania is genus of trypanosome protozoa which is transmitted

by the bite of certain species of sand fly – Phlebotomus Transmitting period – before the main rainy season Reservoir: Animals – dogs ( mainly Europe), fox, rats, jackals…… Most commonly KA is spread human to human, however transmission from animal to human is possible but less common

(Sudan) Others: congenital, needles (drug

abuse), blood transfusion, sexual, bitesfrom infected animal

HANDLING OF LEISHMANIA DONOVANI Viable organism are restricted in a particular area Admission to that room restricted to authorized person After work workers should wash their hand well Appropriate protective clothing (disposable gloves, sky blue

or white laboratory coats) should be worn Cut and scratches should be kept covered Mouth pipetting should not take place Eating, drinking, chewing, smoking, storage of food,

cosmetic use must not take place All west material must be made safe disposal Viable organism must placed in sealed containers and label

appropriately.

OUT LINE OF DRUG DEVELOPMENT AGAINST LEISHMANIA DONOVANI Culture of Leishmania donovani

Culture medium : RPMI (Roswell Park Memorial Institute)-1640 mediumwith L – glutaminewithout sodium bicarbonate(Moore et al., at Roswell Park Memorial Institute)

Culturing of cell : Primary culture:

RPMI culture medium complemented with 10% heat inactive fetal calf serum (i.e 9ml RPMI+1ml FCS for standard 10ml culture)Then inoculate the cell and incubate in an airtight vessel with black cap at 25oC for 4to 5 days.

Sub culturing:every after 4 to 5 days 0.5 ml old culture transferred into new 9.5 ml medium

Testing minimum inhibitory concentration (MIC) of desired drug:after design bioinformatically and prepare synthetically the drug tested in Leishmania donovani.10ml log phase cell culture (0.5ml) transfer into new culture medium (9.5ml) with different drug concentration in different culture tube.after incubation at 25oC which concentration shows IC50 that was selected for further test

Test on animals: Clinical trials

DNA PREPARATION OF LEISHMANIA SP. FOR DNA SEQUENCING 10 ml Leishmania culture Pellet cell by centrifuge for 10 min in 1000rpm Resuspand cell in 0.5 ml TELT (Tris, EDTA, LiCl, Triton x-100) and

add 10 µl RNaseA (10mg/ml) Incubate for 5 mins Add 0.5 ml phenol, vortex, centrifuge at 5,000 rpm for 5 mins Recover the top aquous phase Mix with same volume of chloroform/isoamyl alcohol (24:1) Add chilled ethanol Centrifuged at 14,000 rpm for 30 mins at 4oC Wash the pellet with 70 % ethanol Dry and resuspended in 50 µl TE buffer PCR the desire gene Purified the PCR product and sequencing the DNA

THANK YOU

sitamaquine or kalazaquine Miltefosine (Impavido)