Uploaded on:
Category: Medical / Health
Phases of Commercial Drug Development. Drug Development is a round of wearing down. The Challenge
Slide 1

A Workshop on High Throughput/High Content Screening Applications to Target-based Drug Discovery Research SMALL MOLECULE SCREENS Clifford Stephan, Ph.D. Inquire about Assistant Professor John S. Dunn GCC Chemical Genomics Research Consortium Scott R. Gilbertson, Ph.D. Teacher M.D. Anderson Chair in the College of Natural Sciences and Mathematics Department of Chemistry, University of Houston

Slide 2

Stages of Commercial Drug Development Basic Research Target Validation HTS and Lead Id Lead Opt Preclinical Tox File IND Clinical Phase 0/1 Clinical Phase 2 Sales Marketing Phase 4 Clinical Phase 3 File NDA Approval Drug Development is a session of wearing down. The Challenge … Select 1-2 mixes from the a great many conceivable outcomes that will be protected and adequate in people

Slide 3

Stages of Academic Drug Development HTS and Lead Id Lead Opt Basic Research Still a round of whittling down. The Challenge … Identify operators that expansion the basic logical information for a specific focus with the likelihood of giving further approval of the objective as a "druggable" target. Hold the likelihood of recognizing a lead arrangement of mixes that could take our examination in new, startling bearings. The likelihood of setting up protected innovation and the reason for a future pharmaceutical.

Slide 4

Why perform High Throughput Screening? HTS empowers the testing of expansive quantities of compound substances for movement in various regions of science in a moderately brief time. The whole substance space of little natural atoms is evaluated to be > 10 60 . Of those, ~ 2.7 x 10 7 mixes have been enlisted and made. (Nature Insight, 2004) Responses contemplated can run from biochemical frameworks of filtered proteins or compounds to flag transduction pathways to complex cell (Systems Biology).

Slide 5

High Throughput Screening: A relative term HTS in Pharma and Biotech is a procedure inexactly characterized as testing 10,000 to 100,000 information focuses/day utilizing "industrialized" techniques UltraHTS test >1,000,000 information focuses/day HTS in the Dunn Screening Core the capability of screening 100\'s to >10,000 information focuses/day taking after Pharma industry guidelines

Slide 6

The Screening Continuum From a publication by RR Tice et al of the National Toxicology Program HTS Initiative, 2007

Slide 7

Classes of HTS Assays Homogeneous \'Mix and Read" style examines Simple with expansion steps just, higher throughput Examples: Cell suitability Live cell imaging Proximity (e.g., radioisotope, FRET, ALPHA) Enzyme Kinetics Heterogeneous Traditional style tests Multiple steps, more controls, slower throughput Examples: Traditional restricting measures Traditional sandwich ELISA

Slide 8

Compare Traditional Assays with HTS Assays

Slide 9

Key components for fruitful HTS time/well wells/day screens/year extend Time HTS Screen Quality Costs couple of false positives couple of false negatives S:N,SW,z\'- Factor Validated "Hits" reagents consumables instrumentation faculty

Slide 10

HTS: An Iterative Process Research bunches Target Id and Validation Develop Primary and Secondary examines Define criteria for dynamic mixes Direct "Hit" change prepare HTS Group Perform Primary Screen Purpose: Identify a beginning spot Method: Interrogate libraries of mixes/qualities HTS Group Secondary Screen Purpose: Validate introductory "Hits" Method: Selection of mixes or restorative science Chemistry bunches Analysis and understanding of Data for Structure Activity Relationships Refine and enhance recognized "Hits" Modeling and therapeutic science Selection of mixes for screening by means of virtual screening, centered libraries

Slide 11

Critical Issues to be Addressed Prior to Testing the First Compound Key variables that must be tended to before screening: Assay convention (scaling down/improvement) DMSO resistance (test 0.1 - 5%), standard compound vehicle Reagent amount and cluster consistency Reagent solidness for capacity and use under test conditions Appropriate positive and negative controls Assay reproducibility and flag soundness Available optional or counter screen to test target specificity and selectivity

Slide 12

Consider: Reagent Quantity Compare customary measures with HTS tests

Slide 13

Consider: Reagent Stability Compare customary tests with HTS measures Traditional test: Reagent strength 30-minutes to physically setup plate 2-hr hatching for an end-guide estimation 5-minutes toward read plate Reagents should be steady for up to ~2.5hr HTS test: Reagent dependability 5-minutes to setup every plate 2-hr brooding for an end-direct estimation 5-minutes toward read plate, 40 plates/run add up to Reagents should be steady from first to last plate Up to ~3 hrs for plate setup, ~3hr read time Up to 8 hrs all the way Can remaining reagents be reused

Slide 14

Consider: Availability of fitting positive and negative controls Traditional appraisals of test quality S/B = Mean PosCtl/Mean NegCtl S/N = (Mean PosCtl - Mean NegCtl)/StdDev NegCtl Assay 1 Ve+ mean 50, Ve-mean 10 S/B = 5, S/N = 13 Assay 2 Ve+ mean 112, Ve-mean 10 S/B = 11, S/N = 39

Slide 15

Availability of proper positive and negative controls Common HTS evaluation of test quality (3 * StdDev PosCtl) + ( 3 * StdDev NegCtl) z\' = 1 - - - - Mean PosCtl - Mean NegCtl

Slide 16

Availability of suitable positive and negative controls Common HTS appraisal of test quality Separation Band Ve+ mean 50, Ve-mean 10 S/B = 5, S/N = 13, z\' = 0.5

Slide 17

Availability of suitable positive and negative controls Common HTS evaluation of test quality Ve+ mean 112, Ve-mean 10 S/B = 11, S/N = 39, z\' = 0.0

Slide 18

Availability of suitable positive and negative controls Common HTS appraisal of test quality z\' = 0.5 z\' = 0.1

Slide 19

Assay Issues to be Addressed Prior to Testing the First Compound Plate Uniformity and Signal Variability Testing Critical testing of a test framework preceding screening for all tests keep running in the center These examines test the execution of the accompanying controls: Maximum flag reference (most noteworthy test end point) Minimum flag reference (foundation/least test end point) Midrange flag reference (flag changeability appraisal)

Slide 20

Assay Issues to be Addressed Prior to Testing the First Compound For all tests keep running in the center, comparative outcomes must be acquired more than three separate days (autonomous investigations in triplicate) utilizing all gear and compound vehicle that will be utilized amid the screen. Acknowledgment criteria: Intraplate changeability: No obvious edge impacts or float %CVmax and %CVmin < 20% z\' ≥ 0.4 Interplate and Inter-Day inconstancy: Midrange control < 2-overlay inside a solitary day Midrange control < 2-crease over any two days

Slide 21

What Are These Small Molecules We Test? They are not DNA, RNA, or protein macromolecules Practical Definition: An natural atom of under 1000 Daltons Typically in the scope of 300-700 Daltons Small natural particles made by living creatures (e.g., common items) Small natural particles made by scientific experts (e.g., natural mixes, RNAi) In all cases one is searching for a little \'medication like\' natural molecule that shows an organic action (e.g., agonist, rival) with the objective of intrigue.

Slide 22

How Does One Select a Library to Screen? Arbitrary Selection Random high throughput screening Little is thought about the objective Few or no dynamic mixes as aides Computational Chemistry/Virtual Screening Creation of \'Centered Libraries\' Requires earlier information about target Active mixes, 3D-Structure Sequence homology Prior Experience Library effectively utilized for comparable or related targets

Slide 23

Core Lab HTS Hit Guidelines On every screening day, z\'- calculate (controls) is assessed for each plate Controls must meet unique acknowledgment criteria previously characterized for the test Only anomalies dropped are those made in light of the fact that of test mistake or those > 3 SD from mean for all of that specific control No more than 10-25% of a specific control will be dropped for a specific plate z\' = 0.5 z\' = 0.1

Slide 24

Core Lab HTS Hit Guidelines Active mixes are those outside 3 SD from the mean for all test specialists on a legitimate plate. On the off chance that recreates are played out, a test operator must be dynamic on ≥ 66% of all repeats to be named dynamic. "Hits" are those dynamic intensifies that show concentration reaction upon reorder and retest.

Slide 25

Helpful References: Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS. High-throughput screening measures for the distinguishing proof of synthetic tests. Nat Chem Biol. 2007 Aug;3(8):466-79 . Zhang JH, Chung TD, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen. 1999;4(2):67-73 . Iversen PW, Eastwood BJ, Sittampalam GS, Cox KL. An examination of test execution measures in screening examines: flag window, Z\' element, and test fluctuation proportion. J Biomol Screen. 2006 Apr;11(3):247-52 . Inglese J, Shamu CE, Guy RK. Reporting information from high-throughput screening of little atom libraries. Nat Chem Biol. 2007 Aug;3(8):438-41 . National Institutes of Health (NIH) Chemical Genomics Center (NCGC), an individual from the Molecular Libraries Probe Production Center Network.

Slide 26

Cliff Stephan, Ph.D. Postdoctoral preparing Cardiovascular Division B.A. Science and Molecular Biology Ph.D. Pharmacology Research Instructor, Cardiovascular Department, Hypertension Division Director of High Throughput Screening Head of the John S Dunn Central Scree

View more...