drug development and out line of drug invention against Leishmania donovani

1. drug development and out line of drug invention against Leishmaniadonovani Biomadicalinfomatics center Rajendra Memorial Research Institute of Medical Science (RMRIMS) Moonmoon Deb Ph.D scholar NIT Rourkela Life Science Department 509LS102

2. Index • Drug discovery • Protein homology modeling • Leishmania sp. and Kala-azar • Out line of drug development against Leishmaniadonovani • DNA isolation from Leishmaniadonovani

3. 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.

4. 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

5. The Drug Discovery Process(Modern Methods of Drug Discovery) • Drug Target Identification • Target Validation • Lead Compound Identification • Lead Optimization • Preclinical and Clinical Development

6. 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) bioinformatics c) proteomics

7. 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.

8. 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.

9. 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

10. 3) Lead Compound Identification • Compounds are mainly identified using random (screening) or rational (design) approaches.

11. Traditional Methods of Drug Discovery natural (plant-derived) treatment for illness/ailments ↓ isolation of active compound (small, organic) synthesis of compound ↓ manipulation of structure to get better drug (greater efficacy, fewer side effects)

12. 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 )

13. Ligand database Target Protein Molecular docking Ligand docked into protein’s active site

14. 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.

15. 5) Clinical Trials: Phase I: Single Ascending Dose (SAD) studies Multiple Ascending Dose (MAD) studies Food effect 80% of drugs fail the Phase I clinical trial. Phase II Phase III

16. 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

17. 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

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

19. Tertiary structure prediction Homology modeling SWISS-MODEL  - An automated knowledge-based protein modelling server 3Djigsaw - Three-dimensional models for proteins based on homologues of known structure CPHmodels - Automated neural-network based protein modelling server ESyPred3D - Automated homology modeling program using neural networks Geno3d - Automatic modelling of protein three-dimensional structure SDSC1 - Protein Structure Homology Modeling Server ThreadingPhyre (Successor of 3D-PSSM) - Automated 3D model building using profile-profile matching and secondary structure Fugue - Sequence-structure homology recognition HHpred - Protein homology detection and structure prediction by HMM-HMM comparison Libellula - Neural network approach to evaluate fold recognition results LOOPP - Sequence to sequence, sequence to structure, and structure to structure alignment SAM-T02 - HMM-based Protein Structure Prediction Threader - Protein fold recognition SWEET - Constructing 3D models of saccharides from their sequences AbinitioHMMSTR/Rosetta - Prediction of protein structure from sequence

20. Then click on SWISS-MODEL • Then click on automated model • Email ID • Project title and copy pest the sequence • Discovery studio

21. Leishmania sp. and Kala-azar • Leishmania sp. is responsible for KALA-AZAR (KA) or Leishmaniasis • Leishmaniais 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, bites from infected animal

22. Handling of Leishmaniadonovani • 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.

23. Out line of drug development against Leishmaniadonovani • Culture of Leishmaniadonovani Culture medium : RPMI (Roswell Park Memorial Institute)-1640 medium with L – glutamine without 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.

24. 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 Leishmaniadonovani. 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

25. 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

26. Thank you

27. sitamaquine or kalazaquine • Miltefosine (Impavido)