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Fragment based drug design complementary tool for drug design

fragment based drug design is complementary computer aided technique to analysis and study different molecule on protein binding receptor etc.

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Fragment based drug design complementary tool for drug design

  1. 1. FRAGMENT BASED DRUG DESIGN- COMLEMENTORY TOOL FOR DRUG DESIGN GORGILE AMOL T. M.S.(MEDICINAL CHEMISTRY) MC/2014/08 NIPER HEDERABAD
  2. 2. CONTENT • Introduction • Concept and review • Method for fragment screening • Case study • Advantageous • Conclusion • Reference
  3. 3. INTRODUCTION Historical development- • Abbott laboratories pioneered in the introduction of SAR-FBDD by using NMR, in 1990. • Followed by HTS crystallography in 2000. • So, it examine 23 protein by SAR-NMR method with 0-0.9% hit rate for various sampling. • They were identify potent <300 µM inhibitor. • The late 1990s the method was developed as HTL (hit- to- lead). • ADVANTAGES OF FBBD-  Smaller screning libraries  Higher hit rates  Improve physicochemical properties  Opportunities for chemical novelty
  4. 4. Concept and overview- What is FBBD • “The construction of smaller , low MW, less complex molecular structure that presnt only limited no. of pharmacophores and degrees of conformational freedom” also known as scaffold or templet. • FBDD based on FBDD based on Lipinskis rule-5 not > 5H bond donar, not > 10 H bond accepter, Clog P< 5, MW < 500. Rule of -3 molecular weight < 300 Da cLogP < 3,No.of H bond donors /accepter < 3 Fig : FBDD workflow
  5. 5. Fragmentation pattern
  6. 6. METHOD FOR FRAGMENT SCREENING A) IDENTIFICATION OF FRAGMENT HIT- B) FRAGMENT OPTIMIZATION- 1. NMR- it provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules. NMR PROTEIN DETECTED NMR MODERATE AFFINITY BINDER Kd- 100µM HIGH AFFINITY BINDER Kd -19µM LIGAND DETECTED NMR SATURATION TRANSFER DIFFERENCE- NMR (STD-NMR) WATER LIGAND OPTIMIZED GRADIENT NMR (LOGSY) FLUORINE CHEMICAL SHIFT ANISOTROPHY AND EXCHANGE FOR SCREENING (FAXS) TARGET IMMOBILISED NMR( TINS)
  7. 7. 2. X- ray crystallography- Detection of hit fragment by Cocktail method- Astex Therapeutics identifie fragments for the cyclin dependent kinase (CDK) 2 AT7519 and AT9283 Akinase inhibitor for cancer therapy. 3.Surface Plasmon Resonance- SPR to identify potent fragment hits against BACE-1, Pim-1, HIV-1 reverse transcriptase, HIV-1 protease, carbonic anhydrase II, human serum albumin, thrombin, chymase .
  8. 8. 4. Biolayer interferometry- BLI it measures changes in the interference pattern of light between the sensor and the solution. 5. Isothermal Titration Calorimetry- ITC is a thermodynamic technique that measure the heat released or absorbed during a biomolecular binding event. It also determination of thermodynamic properties like – Binding constants (KB), reaction stoichiometry (n), enthalpy (ΔH) & entropy (ΔS).
  9. 9. 6. MASS SPECTROMETRY-ESI-MS NON COVALENTLY BOUND FRAGMENT Kd upto mM range COVALENTLY BOUND FRAGMENT A French company NovAliX They screened a fragment library of about 350 compounds against Hsp90 which resulted in 40 fragments binding to Hsp90. 7. Weak affinity chromatography- this technique allows the detection of fragments in the 1mM to 10μM range.
  10. 10. 7. Capilarry electrophoresis- discovery for multipal compound 8. Ultrafiltration- (affinity based separation )of bound and unbound fragment The screening resulted into 3 and 9 fragment hits for riboflavin kinase and methionine aminopeptidase 1 respectively. 9. Biochemical assay/ high concentration screening- by this method good hit fragment can identify , high concentration required high solubility
  11. 11. Strength and Weaknesses of Some of Commonly Used Experimental Fragment Screening Methods Screening method Throughput Protein requirement Sensitivity Advantages Disadvantages Ligand detected NMR 1000s Medium-high (µm range) 100nM-10mM High sensitive not required labelled protein expensive, false +ve rate is high, cannot detect tight binders Protein detected NMR 100s High (50-200mg) 100nM-10mM Provided 3D structure information strument is expensive, require isotope labeled protein, expert required X-ray crytallography 100s High (10-50mg) 100nM-10mM Provided detail 3D structure information Expensive, well diffracted high crystal require SPR 1000s Low (5µg) 1nM-100mM Provide kinetic data association dossociation rate,kd,kb Protein immobilization on gold surface requird ITC 10s Low (50-100µg) 1nM -1mM Provide high quantitative affinity data Require high sample cocentration MS 1000s Low (few µg) 10nM-1mM No need protein immobilization Require choice of buffer, aggregation Biochemical assay >10000 Low (<100µg) Not available Simple method Reqire knowledge of biochemical function
  12. 12. Fragment optimization • Fragment growing is the stepwise addition of functional groups or substituents to the fragment core to maximize the favorable interactions with the binding site residues • The fragment linking approach is based on covalently linking two or more fragments bound independently in proximity with suitable linkers.
  13. 13. CASE STUDY 1. Fragment screening against HIV -1 protease • HIV-1 protease is a dimeric aspartic acid protease. • Screening- consist of 384 fragment (MW142 Da) ,screening carried out by two method 1. soaking- in that crystal p41 (pdb_id : 2PCO) grow in the MgCl2 medium (is not compatible with DMSO) individual crystal soaked at active site 10% DMSO, were done with C2221 crystal as monomer Data was collected at 7% of compound but no HIT observed. So, another crystal form P21212,(pdb_id: 3E43) used further soaking with mix. Of 4 compound(2.5mM) ,(10 min- 1hr ) data collected of 108 crystal (17%) fragment hit observed 2. cocrystallization- The P6122 crystal(pdb_id: 3KFP) grow and diffracted to 1.8- 2.5Å IN 10% DMSO Containing 4 comp.at 50mM data collected containing 160 crystal at Fig. Overview of the HIV life cycle
  14. 14. Description of hit In fig. having two binding site , an overall hit rate of 0.8% (1F1) ,(2F4) bind in the flap site of HIV protease, and 2- methylcyclohexanol (4D9) binds in the exosite of HIV protease. Fig. A 1. Binding of 1F1 in the flap site of HIV-1 protease. B.1. Binding of 4D9 in the exosite of HIV-1 protease. A.2Fragment binding sites on HIV-1 protease. B.2 Chemical structures of compounds for fragment screening against HIV-1 protease.
  15. 15. 2. Fragment screening against HIV-1 reverse transcriptase-  HIV-1 reverse transcriptase as a drug target.  It is a the most important drug target for HIV.  RT is a heterodimer of p66 and p51 subunits, with four polymerase subdomains. Fig. RT-rilpivirine complex shown as a cartoon. Rilpivirine (brown space filling) is bound at the NNRTI-binding pocket. The p66 subdomains are color-coded fingers Ordered waters are shown as blue dots, and DMSO-d6 molecules are shown as green, yellow, and red spheres.
  16. 16. Fragment screening by SPR • In the primary screen, 1040 fragments were screened individually for binding to RT via SPR. • Compound were screened at 4 concentrations from 50 to 400 mM. • Fragments show KD value of <1 mM and stochiometries for binding of 0.75-5 times that observed for the nevirapine. • An about 40% of the initial hits were discarded on the basis of undesirable interaction of fragment. slow dissociation • so, molecule it has been rejected as hit . • After removing those fragments with undesirable SPR profiles, • 96 compounds were selected for further experiments.
  17. 17. competition and inhibition- • After the screening of 96 compound to select only those compound which bind on NNRTI pocket . • The fragment were at 200 µM and nevirapine 20µM, • 10 compound which has been screened further, from that 2 of the fragment found false +ve , while remaining show inhibitory activity . • Only one fragment was found that inhibited all variants of RT tested, A, 4-bromo- 1-indanone.
  18. 18. X-ray crystallography-based fragment screening against RT: novel druggable pockets • HIV-1 reverse transcriptase is a highly dynamic protein, and • motions in the interdomain hinges are thought to be critical to its function. • A fragment based screen by X-ray crystallography against an NNRTI inhibited RT uncovered 16 sites on the protein that bind fragments, several of which represent novel druggable targets, Fig. Fragment binding sites in reverse transcriptase. Fragments are shown as orange spheres. Rilpivirine is shown as yellow spheres. The p66 subunit is in dark green and the p51 subunit in cyan. Fragment binding sites that are described are circled.
  19. 19. Library design, soaking optimization, and data collection  Crystal RT52A rilpivirine is used for soaking, it tolerate DMSO.  Soaked in 5% ehylene glycol (for cryoprotection).  And 20% v/v DMSO 20mM for cocktail crystal(1-2 min).  No of comp,bind to RT in the presence of 80mM L-arginine.  It can also observed by trimethylamine N-oxide 6%v/v.  And improved resolution of crystal from 1.8-1.5Å  And compare the DMSO with high resolution .  Fragment binding was observed.
  20. 20. Soaking results: numerous allosteric binding sites • The 742 fragments soaked, 34 hits were obtained, for a hit rate of 4.4%. • These 34 compounds bound to 16 different sites of RT
  21. 21. CONCLUSION • Fragment based drug design (FBDD) is a powerful and widely used drug discovery approach. • It involves the identification of low molecular weight chemical fragments and their optimization into lead compounds. • The generating high quality lead compounds for a variety of targets. • The success in fragment based drug discovery use of recent technological advancements in fragment screening technologies. • Computational methods have already played important roles both in selecting the initial fragments. • Construction and deconstruction approach • In novel drug development.
  22. 22. Recent literature • Use of FBDD in the Discovery of Two Series of Potent Methionine Aminopeptidase-2 Inhibitors Chris McBride, Staff Scientist, Medicinal Chemistry, Takeda This presentation will demonstrate the strategy used to identify two series of methionine aminopeptidase-2 (MetAP2) inhibitors. Fragment libraries were screened for hits with high ligand-efficiency (LE) and orthogonal hit confirmation led to a low affinity indazole core being selected for directed elaboration with the aid of structural information. Additionally, structural insight and SAR from the indazole series led to the design and accelerated knowledge-based fragment growth of the pyrazolo[4,3-b]indoles as MetAP2 inhibitors. • FBDD on Metalloprotein Targets Seth M. Cohen, Ph.D., Professor, Department of Chemistry and Biochemistry, University of California, San Diego Most inhibitors of alloproteins employ a functional group that binds to the active site metal ion. We have developed an FBDD approach to metalloenzyme inhibitors by developing a library of suitable metal-binding pharmacophores (MBPs). Thermodynamic and structural investigations are being used to provide insight into the influence of the protein active site on MBP binding. In this presentation, our efforts on utilizing these MBPs for metalloprotein inhibition wil • Advances in SPR Fragment Screening by Andrew L. Hopkins, DPhil, SULSA Research Professor, Translational Biology, University of Dundee SPR has become the workhorse method for rapid and accurate fragment screening. We present results from a collaborative effort between the Hopkins-Navratilova lab and GE Healthcare prototyping new assays methods and new technology to advance SPR fragment screening. We will introduce the first results on a new, prototype Biacore™ instrument.l be discussed. • Using Fragment- Based Lead Discovery towards Alternate Mechanisms: Inhibiting Ubiquitin Binding to USP7 Till Maurer, Ph.D., Senior Scientist, Structural Biology, Genentech Small molecule inhibitors targeting the deubiquitinase Ubiquiti Specific Protease 7 (USP7) have potential as cancer therapeutics. Using ligand-based NMR in an activity agnostic FBLD effort, we have identified binders to several sites in USP7 including a unique site in the “palm” of USP7. These could be shown to be active. The palm series imply a very distinct mechanism of action independent of the catalytic triad by binding in a region involved in USP7-Ubiquitin interaction.
  23. 23. References • Bennett T. Farmer and Allen B. Reitz, Fragment based drug discovery, Wermuth’s The Practice of Medicinal Chemistry, Elsevier Ltd 2008, 228-243. • Theresa Tiefenbrunn*, C. David Stout, Towards novel therapeutics for HIV through fragment-based screening and drug design. Published by Elsevier Ltd. Progress in Biophysics and Molecular Biology 116 (2014) 124e140, 2014. • Christopher W Murray1 and Tom L Blundell2, Structural biology in fragment-based drug design, www.sciencedirect.com,Current Opinion in Structural Biology 2010, 20:497–507. • A. Kumar, A. Voet and K.Y.J. Zhang* Fragment Based Drug Design: From Experimental to Computational Approaches, Bentham Science Publishers, Current Medicinal Chemistry, 2012, 19, 5128-5147. • James Lanter, Xuqing Zhang, and Zhihua Sui, Medicinal Chemistry Inspired Fragment-Based Drug Discovery, Methods in Enzymology, Volume 493, 2011 Elsevier Inc. • Renee L. DesJarlais Contents, Using Computational Techniques in Fragment-Based Drug Discovery, Methods in Enzymology, Volume 493, 2011 Elsevier Inc. • Jianguo Li a,b,g, Shouping Liu a,g, Jun-Jie Koh a,e, Hanxun Zoua, Rajamani Lakshminarayanan a, Yang Bai a,c, 4 Konstantin Pervushin c, Lei Zhou a, Chandra Verma a,b,c,d, RogerW. Beuerman a,e,f,g, A novel fragment based strategy for membrane active antimicrobials 2 against MRSA, Published by Elsevier B.V., Biochimica et Biophysica Acta xxx (2015) xxx–xxx • http:// practicalfragments. blogspot.com • http://fbdd-lit.blogspot.com
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fragment based drug design is complementary computer aided technique to analysis and study different molecule on protein binding receptor etc.

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