Química Medicinal

HTS & HCS

Prof. Dr. Andrei Leitão

 HTS & HCS

HTS: ensaio em massa (High-throughput assay)

HCS: ensaio de elevado conteúdo (High-content screening), também

denominado HCA (High-content analysis) → baseado em imagem

https://www.genengnews.com/insights/drug-discovery-approaches-utilizing-three-dimensional-cell-culture/ 2
 HTS setup

Chapter 5. High-Throughput and High-Content

Screening for Huntington’s Disease Therapeutics
Hemant Varma, Donald C. Lo, and Brent R. Stockwell.

In: Neurobiology of Huntington's Disease:

Applications to Drug Discovery.

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 HTS setup

https://www.gesundheitsindustrie-bw.de/en/article/press-
release/hit-discovery-constance-gmbh-a-new-hub-for-hts-and-
compound-management

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HTS & HCS

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HTS & HCS

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HTS & HCS

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 Fastest - first and best
Hit Hit to Lead
Target HTS
Evaluation Lead Optimisation

information

Potency Efficacy DESIGN AND Kinetics

Selectivity SYNTHESIS Metabolism
Enzymology

compounds compounds
Lead
HTS + Combichem
Teague, Leeson, Oprea, Davis, Angew Chem 1999, 38, 3743
 “Universal” Library
160 H
R N
Approach 1 SH
O

R40X
R160CO2H
Solid Solution
phase phase

160 H 40
H2N R R N R
S S
O

Approach 2
STEP 1 H H
R 80 STEP 2 R
80
N
H2N i) Solid phase R80CO2H R N
Br Nu Nu
ii) Cleave O O

Walters and Teague Tet Lett. 2000, 41, 2023

 HTS Screening Hits
• Drug-like hits
• potency of many underperform
• binding via weak non-specific interactions
• not all interactions made or very suboptimal
• would explain “flat SAR” in Hit-to-Lead activities
• small mM leads easier to optimise than large mM
• “easy” and “difficult” hit-to-lead projects
– easy to increase Mwt/logP - increase potency
• easy to demonstrate SAR, increase potency 10x
– difficult because of flat SAR
• difficult to reduce Mwt and logP maintaining
potency
Teague, Leeson, Oprea, Davis, Angew Chem 1999, 38, 3743
Exemplos

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HTS Examples - GPCR Project
acid R
S CONH2

N
IC50 = 0.55 mM
IC50 = 4.6 mM Mwt 350
Mwt 268 clogP 3.7
ClogP 3.4

R H
N

O OH
S
O
IC50 = 0.18 mM
Mwt 380
ClogP = 4.5
 GPCR Hit-to-Lead
R
S CONH2

Many analogues
same or loss potency

Many analogues
same potency

R H
N

O OH
S
O

• Both series dropped -

 GPCR Hit-to-Lead
acid acid

N
Cl
Cl

IC50 = 4.6 mM IC50 = 0.02 mM

Mwt 268 Mwt 336
ClogP 3.4 ClogP 5.3 (:-<)

• Rapid Hit-to-Lead optimisation

– clear SAR
– drug-like series with good DMPK
– patentable
 Summary
• HTS
– starting points are crucial to speed throughout
process
– screening file should reflect what chemists can
easily work upon
– ideally we all want to find drugs in our screening file
• but generally a HTS finds only leads not drugs
– file-size isnt everything = quality is equally important

• Libraries
– Many approaches - targeted libraries v successful
• kinase libraries - 4x hit rate - screening file
– libraries should reflect what you wish to find
• leads not drugs Teague, Leeson, Oprea, Davis, Angew Chem 1999, 38, 3743
Cell-based assay

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Cell-based assay

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 Cell-based assay

✓ 25 000 compounds tested

✓ Cell lines: AR-dependent (LNCaP) and AR-independent (PC3)

✓ Flow cytometry for adherent cells

Cell membrane
Granules

Nucleus

Haynes, M. K.; Leitão, A. et al., J. Biomol. Screen. 2009, 14, 596 18

 Cell-based assay

% response
AR-dependent EC50 = 0.77 μM
Family 1
AR-independent EC50 = 6.5 μM
log [M]

% response

EC50 = 3.35 μM
Family 2
log [M]
Haynes, M. K.; Leitão, A. et al., J. Biomol. Screen. 2009, 14, 596 19
 Always check your system
Negative control
Estradiol
Granulation of breast [cpd] = 50.0 µM
cancer cells (MCF-7) is
observed for Morus alba
extracts high ethanol
proportion
Compound
precipitation
(green and cells
(blue) Ethanol/water (80:20) Ethanol/water (95:5)
[cpd] = 6.25 µM

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Panorama da descoberta de fármacos

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 A academia

Parcerias com a
academia 28 %
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Parcerias...

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 Parcerias...

Nicolau, K.C. Chem. Biol. 2014, 21, 1039 24

Onde estamos?

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 Qual é a estratégia terapêutica?

Fármaco
Biofármaco

http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugInnovation/ucm429873.
htm
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