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1) In 1960-combinatorial chemistry was                           introduced.                        2) The drug discovery ...
A1                    B1           A2                    B2           An                     Bn• The structure of the comp...
Combinatorial                                         synthesis                                                         So...
1. In this method ,the reaction is carried out on a solid   support such as resin beads.2. The solid phase synthesis was p...
A cross linked insoluble polymeric support-resin.An anchor or linker covalently linked to resin.A bond linking the subs...
RESIN        LINKER   FUNCTIONAL                 GROUPBEAD
1.Polystyrene resins2.Tentagel resins3.Pepsyn4.PEGA5.Glass and ceramic beads
The linker is the molecule that site between the compound and the support.The linker moves the point of attachment of su...
WANG RESIN: linker suitableRESIN       LINKER                F   for attachment & release of                              ...
Protecting groups are important for blocking& regenerating certain functional groups in a             reaction sequence.So...
R2               HO                [BOC]                             NO                            H           O          ...
Here the compounds are synthesised in separate vessels but at the same time parallel.The array of reactions are taken ei...
A bath of resin is divided in to equal portion in reactionvessel.Each portion of resin is treated with treated withdiffe...
A           B       C    A           A       A    B           B       B    C           C       CA   A       B   A       A ...
SEALPOLYPROPYLENEMESH RESIN
The reaction vessel consists of brush likearray of pins, at the end of it consists of bead   (lollypop) with suitable link...
Spatial array of microchips , which is    embedded with resin beads
Unlike one bead - one compound synthesis,solution phase synthesis often lead to mixture ofproducts in one pool.Most of t...
R-CO-Cl + RI-NH2         R-CO-NH-RI + HClAcid chloride based set:-A1 + (B1,B2,B3…………B10)        mixture1 containing all po...
R1    COOH          R2            NH2       R3       CHO        NC                         O             R3        H      ...
POOLA subset of a given combinatorial library.The process of combining & mixing librarycomponent.BUILDING BLOCKS: One of...
1)   One bead one compound strategy2)   Iterative deconvolution3)   Subtractive deconvolution4)   Bogus coin deconvolution...
It is the process as a result of which the combinatorialexp becomes less complex.It is usually done by backtracking & re...
The specific quantity of beads are allocatedfor each possible structure in library.ie beadscontains only molecules of the ...
This is same as mix and split technique.At each gp of beads bearing a variety ofcompounds,but a given structure only app...
This is similar to iterative deconvolution butuses negative logic ie eliminate a functionalgroup if activity is absent.T...
Example:chlorine gp is placed in several position ofphenyl ring.1) The entire library is screened to get baseline   activi...
 The term “orthogonal” means perpendicularIn this type of pooling ,we distribute the functionalgroups to be considered i...
A                  B                 C(aa,ab)            (aa,ac)          (ab,ac)  Shows the pharmacological activity     ...
This is a non iterative screening in which a sub setlibrary is created with a single building block fixed at oneposition ...
Encoding/tagging used as a code to indicate whathappened at each step in the synthesis ,thus identify   the structure of m...
Here specific compounds (tags) are used as a code for           the individual step in synthesis.Eg :ss DNA(oligonucleotid...
COLOURED DYE WITH ISOTOPIC LABELLING
A tiny micro chip is added to resin/to solution.As the various reactions are conducted togenerate the product,at each st...
The tags are encapsulated in glass casing which is stable to chemical &synthetic conditions.Each tag code is associated wi...
Here the solid support acts as an eye to view the reaction
The solid support consists of a ceramic chipcovered with a polypropylene-poly styrenepolymer in which barcode pattern is b...
HPLCIR SPECTRANMR SPECTRAMASS SPECTRAUV-VISIBLE SPECTRA
1) Chemically cleave the compounds from the support & filter   off the beads2) The filter the solution to get the product....
HPLC is highly effective method for separation & detection of components
•IR light is transparent to resin beads ,hence we can analysethe resin bead directly without cleaving the product from it....
NMR gives more structural informationsthan IR/UV.The solid support broadens the peak &hence low resolution.Magic angle ...
MASS spectroscopy analysis is highly automated.The measurement is made on resin beads directly.It is the most widely us...
Solution containing compounds is passedthrough electrically charged capillary & they“explore” in to smaller droplets.
The sample is embedded in the solid matrix(2,5 dihydroxybenzoic acid)Bombarded with laserThe sample molecule are vapori...
Colourimetric detection:Wavelenght ranges from 400-800 nmUV SPECTRA ranges from 200-400nm.Principle:Beer’s-Lambert’s law...
In recent years the peptide-protien & peptide-antibodyinteractions have gained importance in the area of autoimmunedisease...
Peptide suffer from disadvantages like poor bioavailability &unfavorable ph.kinetic profile.Thus the focus has shifted t...
O       R1                O       R3                     H                     N        N                         N       ...
O    O               NH2HN        N            O
oIn contrast to biopolymers,synthetic oligomers are stable toproteases & nucleases.oThey may be linear chain molecules lik...
Solid phase chemistry can also synthesise oligosaccharides.Carbohydrate antibiotics including vancomycin& aminoglycoside...
R2             NH-FMOC                                                                       NH2                   O      ...
CO                                          CONH2           NH                 CO   NH                 RCOCl              ...
It involves identification &validation of target & docking toidentify the hit drug.Virtual libraries are created incombi...
Virtual library: a combinatorial library that has nophysical existance,it exists in computer & can begenerated automatica...
CADD is a combination ofcomputational chemistry andinformation technology tools that helpus to discover new therapeuticsol...
Combinatorial synthesis is now being used in leadoptimsation program of many potent drugs of naturalorigin.Eg:Rhodacyani...
R            S           SN    S                       OHC   N                N        O           R             R1
Nucleoside analogue are useful asantimetabolites & treatment of cancer.By using combinatorial approach,thesynthesis of s...
R1                                N                R1                   N R                                               ...
Automated ,microprocessor controlled robotic process called as HTS.
HIGH THROUHGPUT SCREENINGAutomated microprocessor controlled robotic processHTS : 50000-100000 compounds can be screened...
   CHEMICAL LIBRARY   HTS ASSAYS   INFORMATICS AND ANALYSIS   PROCESS ENGINEERING
MICROPLATE TECHNOLOGIESa.96 well microtiter plate        8 rows (A-H)        12 columns        88 test samples        8 co...
c. 1536 well microtiter plate       32 rows ( A-AF)       48 columns       1,408 test samples       128 controls
IN VITRO MARTIX –PHARMACODYNAMIC         LIGAND     STUDIES         INTERACTION                        STUDIESPHARMACOKINE...
Here an effective target is identified & validatedfor its function.HIT: it is a molecule with confirmed activityfrom pri...
Fl.correlation                spectroscopyScintillation proximity                       Fl.anisotropy assay(SPA)   Surface...
This procedure is suitable to detect receptor          ligand binding reactions.
The molecular interactions of drug & the receptorwill give rise to measurable fluctuations in FI.
When two different chromophores(drug & receptor) interact via  dipole-dipole mechanism, transfers excitation energy non   ...
The sample is illuminated with pulsed laser & the life time of fl.probe is determinedfrom the phase shift between themodul...
The membrane receptor can be immobilised usingaffinity tagsThus flourescence labelled ligand binds with it thenthe recep...
An antibody or a receptor molecule, which is bound to a beademits light when beta emission from an isotope occurs in close...
Absorption studies: caco(coloncancer)cell line grow confluently & form a monolayer onpolycarbonate support or collagen coa...
Due to development of 2-D multi parallel HPLC(SEPBOX,SEPIAtec,Germany) it is now possibleto load up to 5g of NP extract & ...
SEPBOX system works by using gradient elution & polarity basedtrapping in solid phase extraction(SPE).SEPBOX is coupled ...
1. Modern methods of drug discovery by   A.Hillisch and R.Hilgenfeld page no: 72-982. Foye’s Medicinal chemistry by Thomas...
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
Combinatorial chemistry - HTS and its applications in drug discovery
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Combinatorial chemistry - HTS and its applications in drug discovery

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it is my seminar of m.pharm(pharmaceutical chemistry)
on "combinatorial chemistry and HTS and its application in drug discovery "in subject "advanced organic chemistry" for internal assessment.u can also veiw in slideshare ...aryajithu ID

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Combinatorial chemistry - HTS and its applications in drug discovery

  1. 1. 1) In 1960-combinatorial chemistry was introduced. 2) The drug discovery process become highly parallel. 3) Even thousands of structures could be synthesised one at a time. 4) Interestingly biologist introduced HTS to perform invitro assays. In 1963,combinatorial synthesis of peptide on resin bead was done by Bruce Merrifield. It is a tool which allows large no of compounds to be synthesized simultaneously in a time taken to prepare only handful of compounds by traditional synthesis. A chosen set of building blocks are reacted together to make a collection of products known as “library” or “an array”.
  2. 2. A1 B1 A2 B2 An Bn• The structure of the compounds in the mixture are not known with certainty• They are not separated instead each mixture is tested for biological activity as a whole & if active the mixture is analyzed for active compound.
  3. 3. Combinatorial synthesis Solution phase Solid phase synthesis synthesis Parallel Mix & split Tea bag Multi-pin Laminar microchip-synthesis technique synthesis synthesis solid phase spacial array
  4. 4. 1. In this method ,the reaction is carried out on a solid support such as resin beads.2. The solid phase synthesis was pioneered by Merrifield –synthesis of peptides. The bead is treated with different starting materials which bound together. Then it is mixed with another reagent to get the product which is bound to solid support. The excess reagent or by product can be easily removed by washing with appropriate solvent.
  5. 5. A cross linked insoluble polymeric support-resin.An anchor or linker covalently linked to resin.A bond linking the substrate to linker which will be stable to reaction condition.Chemical protecting groups for protecting the functional group not involved in the synthesis.
  6. 6. RESIN LINKER FUNCTIONAL GROUPBEAD
  7. 7. 1.Polystyrene resins2.Tentagel resins3.Pepsyn4.PEGA5.Glass and ceramic beads
  8. 8. The linker is the molecule that site between the compound and the support.The linker moves the point of attachment of substrate away from the surface of the bead.Different linkers are used depending on the functional group(which is present on the substrate) functional group(which is desired on the final product once it is released)
  9. 9. WANG RESIN: linker suitableRESIN LINKER F for attachment & release of carboxylic acids. OH ORESIN LINKER F MERRIFIELD RESIN : linker suitable for peptide products. O O RINK RESIN : Linker suitableRESIN LINKER F for attachment & release of carboxamide. NH2 O OMe MeO
  10. 10. Protecting groups are important for blocking& regenerating certain functional groups in a reaction sequence.Some example of protecting groups are FMOC(fluoro methoxy carbonyl) TBOC(tertiary butyloxy carbonyl)
  11. 11. R2 HO [BOC] NO H O R2 O H NH 2 N [BOC] N H R1 R1 O peptide F3C-COOH N-Et3 O R2 H N [REPEAT] NH 2 R1 O
  12. 12. Here the compounds are synthesised in separate vessels but at the same time parallel.The array of reactions are taken either in grid well in a plastic plate(in bead method) or pins(grid of plastic rods) called crowns.The building blocks are attached to these beads or crowns.The structure of product is identified from the grid code.
  13. 13. A bath of resin is divided in to equal portion in reactionvessel.Each portion of resin is treated with treated withdifferent derivative of first block(A,B & C).After washing the beads are pooled together in one pot& mixed thoroughly.Then split in to equal portion again for coupling to thenext portion.This process is continued until the required library issynthesised.
  14. 14. A B C A A A B B B C C CA A B A A B B B B C B C C
  15. 15. SEALPOLYPROPYLENEMESH RESIN
  16. 16. The reaction vessel consists of brush likearray of pins, at the end of it consists of bead (lollypop) with suitable linker. Here the synthesis takes place. It is inserted in to the plates where thereagents & the solvents kept ,and continously changed.
  17. 17. Spatial array of microchips , which is embedded with resin beads
  18. 18. Unlike one bead - one compound synthesis,solution phase synthesis often lead to mixture ofproducts in one pool.Most of the org reaction occurs in solution phase .For this reason there has been much interest insolution phase synthesis.The main problem here is the difficulty of removingunwanted impurities at each step in synthesis.
  19. 19. R-CO-Cl + RI-NH2 R-CO-NH-RI + HClAcid chloride based set:-A1 + (B1,B2,B3…………B10) mixture1 containing all possible A1-B compoundsA2 + (B1,B2,B3…………B10) mixture2 containing all possible A2-B compoundsA10+(B1,B2,B3…….B10) mixture10 containing all possible A10-B compoundsAmine based set:-B1 + (A1,A2,A3…………A10) mixture1 containing all possible B1-A compoundsB2 + (A1,A2,A3…………A10) mixture2 containing all possible B2-A compoundsB10+(A1,A2,A3…….A10) mixture10 containing all possible B10-Acompounds
  20. 20. R1 COOH R2 NH2 R3 CHO NC O R3 H N R1 N R2 O OH O R3 R5 R4 R2OR OH N R3 O R1 N N R3 OR R1 R2 N R1 R2 O R2 O
  21. 21. POOLA subset of a given combinatorial library.The process of combining & mixing librarycomponent.BUILDING BLOCKS: One of a set ofinterchangable reagents that can be used insynthesis of library.DECONVOLUTIONIsolation & identification of the most activecompound in a mixture is known asdeconvolution.
  22. 22. 1) One bead one compound strategy2) Iterative deconvolution3) Subtractive deconvolution4) Bogus coin deconvolution5) Orthogonal pooling6) Positional scanning
  23. 23. It is the process as a result of which the combinatorialexp becomes less complex.It is usually done by backtracking & reanalysing orresynthesising a subset of structures in the library.The goal of deconvolution is to determine which ofthe mixture of compounds is actually responsible forthe activity.Eg: micromanipulation & sequential release.
  24. 24. The specific quantity of beads are allocatedfor each possible structure in library.ie beadscontains only molecules of the librarymember. They may be tagged. The advantage of this is simplicity ofanalysis & screening.Advances in robotics & automation reducesproblems in this strategy.
  25. 25. This is same as mix and split technique.At each gp of beads bearing a variety ofcompounds,but a given structure only appears in onegroup.In this a small library of structures selected &screened.We find activity in the middle gp of beads ieBAC,BBC,BCC….This tells us that in position ‘1’,B isessential for the activity.The final step is to synthesise this beadedcompounds,keeping then separate & screened each &find out ‘BAC’ IS THE ACTIVE ONE
  26. 26. This is similar to iterative deconvolution butuses negative logic ie eliminate a functionalgroup if activity is absent.Thus functional group that is missing must beneeded for activity.It is useful for “QSAR” studies.
  27. 27. Example:chlorine gp is placed in several position ofphenyl ring.1) The entire library is screened to get baseline activity level.2) If activity is detected,a set of sub library is prepared,each with subtraction of functional group & screened to identify most important functional group.3) The reduced library contains only these functional gp,these are screened to get the active compound.
  28. 28.  The term “orthogonal” means perpendicularIn this type of pooling ,we distribute the functionalgroups to be considered in to a set of sublibraries, A,B,C…etc. which can contain mixture ofsame compounds also.But the functional groups distributed such that anysubset in A & B shares only one functional gp. eg: if we have a very small library of structures-aa,ab,ac……
  29. 29. A B C(aa,ab) (aa,ac) (ab,ac) Shows the pharmacological activity ‘ab’ is the active compound
  30. 30. This is a non iterative screening in which a sub setlibrary is created with a single building block fixed at oneposition & all other building blocks in other position.Here by selecting the functional group from the mostactive subset at each position ,the most activecompound is over all discovered.
  31. 31. Encoding/tagging used as a code to indicate whathappened at each step in the synthesis ,thus identify the structure of most active library member. TAGGING LASER CHEMICAL ISOTOPIC DYE R.F.TAGGING OPTICAL TAGGING TAGGING ENCODING
  32. 32. Here specific compounds (tags) are used as a code for the individual step in synthesis.Eg :ss DNA(oligonucleotide) 6 bases are used for DNA generic code .For decoding, DNA tag is amplified by PCR. --A—B—C—B—C--etc Library compound --R—S—T—S—T--etc Code compound
  33. 33. COLOURED DYE WITH ISOTOPIC LABELLING
  34. 34. A tiny micro chip is added to resin/to solution.As the various reactions are conducted togenerate the product,at each step a rf signal isstored in microchip.This signal can be recalled to identify thesequence of reaction that generated the product.
  35. 35. The tags are encapsulated in glass casing which is stable to chemical &synthetic conditions.Each tag code is associated with the identity of its library member &detected by data base computer.
  36. 36. Here the solid support acts as an eye to view the reaction
  37. 37. The solid support consists of a ceramic chipcovered with a polypropylene-poly styrenepolymer in which barcode pattern is burned ateach step & is decoded visually with the use ofa microscope.
  38. 38. HPLCIR SPECTRANMR SPECTRAMASS SPECTRAUV-VISIBLE SPECTRA
  39. 39. 1) Chemically cleave the compounds from the support & filter off the beads2) The filter the solution to get the product.3) If the solution contains just a single compound we use • IR • UV • MASS SPECTROPHOTOMETER • FLUORESCENCE • NMR If it contains a mixture we use HPLC
  40. 40. HPLC is highly effective method for separation & detection of components
  41. 41. •IR light is transparent to resin beads ,hence we can analysethe resin bead directly without cleaving the product from it.•FTIR will amplify the very small spectral signal from one ormore beads.•The shape of the beads affect the IR spectra,flattned beadsgives strong signal than spherical beads.
  42. 42. NMR gives more structural informationsthan IR/UV.The solid support broadens the peak &hence low resolution.Magic angle spinning NMR: Here thesample is inserted in to the mag.field at theangle of 550 ,this will reduce the peakbroadening,and has been used to analyseswollen polymer beads directly.
  43. 43. MASS spectroscopy analysis is highly automated.The measurement is made on resin beads directly.It is the most widely used technique in combinatorials.
  44. 44. Solution containing compounds is passedthrough electrically charged capillary & they“explore” in to smaller droplets.
  45. 45. The sample is embedded in the solid matrix(2,5 dihydroxybenzoic acid)Bombarded with laserThe sample molecule are vaporized &ionisedThe analysis is done with the use of time of flightanalyser(TOF) In TOF ions of different mass travels different distance ina specified of time.
  46. 46. Colourimetric detection:Wavelenght ranges from 400-800 nmUV SPECTRA ranges from 200-400nm.Principle:Beer’s-Lambert’s law.Lamda max-used as qualitative aspect
  47. 47. In recent years the peptide-protien & peptide-antibodyinteractions have gained importance in the area of autoimmunediseases.By using combinatorials a library of hexa peptides was generated& screened for B7 antibodies binding with CD-2 T-cell proliferation.Peptides screened for inhibition of T cell for type 1-DM & RA.Certain polypeptide screened for inhibition of kinase & proteasefor AIDS &cancer.
  48. 48. Peptide suffer from disadvantages like poor bioavailability &unfavorable ph.kinetic profile.Thus the focus has shifted to synthetic peptido-mimetics likepeptoids. Peptoids are molecules in which the variation occurs in the attachment of amide nitrogen.Similarity: Difference:Molecular weight <500 Lack peptide H-bond More rational flexibilitySide chain,functional gps More stabilityin same position. Less double bond
  49. 49. O R1 O R3 H N N N H H O R2 O R1 O R3 N N N O R2Peptoids are N substituted glycine with increased biologicalhalf life.
  50. 50. O O NH2HN N O
  51. 51. oIn contrast to biopolymers,synthetic oligomers are stable toproteases & nucleases.oThey may be linear chain molecules like oligo nucleotide(ssDNA & ssRNA) oligo ureases.oA library of 5000 oligo peptoids was generated & screened for7-transmembrane G protien coupled receptor inhibition.certainpeptoids were active at nano mol conc.
  52. 52. Solid phase chemistry can also synthesise oligosaccharides.Carbohydrate antibiotics including vancomycin& aminoglycosides has been target incombinatorial chemistry.ExamplesBauhinia purpurea “lectin” analogues.Erythromycin analogues.Neocarzinostatin analogues.
  53. 53. R2 NH-FMOC NH2 O R2 O HO O R1 R1 2 amino benzophenone Solid support:tenta gel R2 NH2 H O O N O R2 R3 O R1 R1Benzodiazepine
  54. 54. CO CONH2 NH CO NH RCOCl RSO2Cl CH2Cl2 OH OSO2R1H2N OH HN RCONH CO RUses:leukotriene antagonist ,carbapenamantibiotics etc.
  55. 55. It involves identification &validation of target & docking toidentify the hit drug.Virtual libraries are created incombinatorial manner to screenthe molecules.This helps to find out noveldrugs against sp.diseases.
  56. 56. Virtual library: a combinatorial library that has nophysical existance,it exists in computer & can begenerated automatically.They are screened againt “rule of 5” or to be docked byusing molecular docking.
  57. 57. CADD is a combination ofcomputational chemistry andinformation technology tools that helpus to discover new therapeuticsolutions.ADVANTAGEStarget specific & structure based fast and automatic very low cost high success rateTwo types:Ligand based drug designStructure/receptor based drugdesign
  58. 58. Combinatorial synthesis is now being used in leadoptimsation program of many potent drugs of naturalorigin.Eg:Rhodacyanin:It is a natural dye with anti malarial activity. it isconsidered as a lead to develop new anti malarialseffective againt quinine resistant plasmodium.In lead optimisation process,Takasu et al,reported asimple one pot method for synthesis of no ofrhodacyanin analogue with variation in “N” containingring.
  59. 59. R S SN S OHC N N O R R1
  60. 60. Nucleoside analogue are useful asantimetabolites & treatment of cancer.By using combinatorial approach,thesynthesis of several nucleoside has beensynthesised.These are effective againts various cancers.Green Burg et al developed a method ofsynthesis of nucleosides in solid state whichare the building blocks for antisensepolynucleotide.
  61. 61. R1 N R1 N R N O G N N N N NHO OH R O R1 N HO OH N O H R1 N N O R O PYRAMIDINE NUCLEOTIDEHO OH
  62. 62. Automated ,microprocessor controlled robotic process called as HTS.
  63. 63. HIGH THROUHGPUT SCREENINGAutomated microprocessor controlled robotic processHTS : 50000-100000 compounds can be screened per week against biological targetu HTS : 10000-100000 compounds within 24hrsHTS assay system consist of sample wells for handling samples
  64. 64.  CHEMICAL LIBRARY HTS ASSAYS INFORMATICS AND ANALYSIS PROCESS ENGINEERING
  65. 65. MICROPLATE TECHNOLOGIESa.96 well microtiter plate 8 rows (A-H) 12 columns 88 test samples 8 controlsb. 384 well microtiter plate 16 rows (A-P) 24 columns 352 test samples 32 controls
  66. 66. c. 1536 well microtiter plate 32 rows ( A-AF) 48 columns 1,408 test samples 128 controls
  67. 67. IN VITRO MARTIX –PHARMACODYNAMIC LIGAND STUDIES INTERACTION STUDIESPHARMACOKINETIC NATURAL PRODUCT STUDIES ISOLATION METABOLISM DRUG SYNTHESIS STUDIES
  68. 68. Here an effective target is identified & validatedfor its function.HIT: it is a molecule with confirmed activityfrom primary hts assay,with good profile in 20assays & with confirmed structure.LEAD: Lead is explained as a hit series for whichSAR is studied.
  69. 69. Fl.correlation spectroscopyScintillation proximity Fl.anisotropy assay(SPA) Surface Fl.resonance sensitive energyfl.detection transfer Fl.life time imaging microscopy
  70. 70. This procedure is suitable to detect receptor ligand binding reactions.
  71. 71. The molecular interactions of drug & the receptorwill give rise to measurable fluctuations in FI.
  72. 72. When two different chromophores(drug & receptor) interact via dipole-dipole mechanism, transfers excitation energy non radially to acceptor chromophore.
  73. 73. The sample is illuminated with pulsed laser & the life time of fl.probe is determinedfrom the phase shift between themodulation of excitation light & emission offlourescence.Eg: measurement of tyrosine kinase activity.
  74. 74. The membrane receptor can be immobilised usingaffinity tagsThus flourescence labelled ligand binds with it thenthe receptor emits fluorescence.Eg:flourescence antagonist on immobilized 5-HT3areceptor.
  75. 75. An antibody or a receptor molecule, which is bound to a beademits light when beta emission from an isotope occurs in close proximity; ie when a radiolabelled ligand binds to bead with receptor or antibody .
  76. 76. Absorption studies: caco(coloncancer)cell line grow confluently & form a monolayer onpolycarbonate support or collagen coatedpolycarbonate support. These are usedfor permeation studies.
  77. 77. Due to development of 2-D multi parallel HPLC(SEPBOX,SEPIAtec,Germany) it is now possibleto load up to 5g of NP extract & to isolate allcompounds in 70-80% purity with in 24hrs.
  78. 78. SEPBOX system works by using gradient elution & polarity basedtrapping in solid phase extraction(SPE).SEPBOX is coupled with photodiode array detector & lightscattering detector in series enables the identification &quantification of the significant compounds.NP database containing about 10,000 structurally characterizednatural compounds ,is being commercialized using MS & 2-DNMR.
  79. 79. 1. Modern methods of drug discovery by A.Hillisch and R.Hilgenfeld page no: 72-982. Foye’s Medicinal chemistry by Thomas and David page no: 56-433. Medicinal chemistry by K.Illango page no: 389-4074. http://www.sciencedirect.com/science5. http://www.liebertonline.com/loi/adt6. http://www.ingentaconnect.com/content7. http://jbx.sagepub.com8. http://www.nature.com/nmeth/index.html9. http://mli.nih.gov/10. http://pubchem.ncbi.nlm.nih.gov/

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