Limitations & Lessons in the use of X-ray Structural Information in Drug Design Davis A.M. et.al., Drug Discov Today. 2008 Oct;13(19-20):831-41.

PRESENTED BY-

DILIP DARADE

PI/316

M.S. (Pharm)

DEPT.OF PHARMACOINFORMATICS

NIPER, HAJIPUR

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Drug Discovery process

Fail Fast Fail Cheap 2

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Drug discovery through CADD

Structure-based drug design

Ligand-based drug design

CADD

Structure - function relationship

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StructureSequence

Function

Atom coordinates along with its electron density

X- ray crystallography

Protein expression

Protein isolation

Protein purification

Understanding the function

Target identification and validation

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Experimental MethodsX-ray crystallography

>85 percent of the protein structures

Atomic detail of proteins

0.1 nm

electron density of the compound 

crystal structure is necessary

the same protein may crystallize into different crystalloid form. 

NMR Spectroscopy Electron Microscopy

direct determination of secondary structures and especially domain movements 

the resolving power of NMR is less 

The cost of the experimental implementation is high

not for the availability of higher molecular masses 

Cellular architecture shape of large proteins molecules

10 nm

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Number of structures in the PDB from 1972 - 2010. Image courtesy of the RCSB Protein Data Bank.

Num

ber o

f rel

ease

d en

tries

Year7

The protein structure is correct

The structure of the ligand & it’s interactions with the protein are correct

The protein-ligand structure is relevant for drug design

Assumptions

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1. The protein structure is correct

Resolution & experience of crystallographer Resolution high- modelling of structure having quality

Electron density

https://cdn.rcsb.org

High resolution <1.5 Å Low resolution >2.5 Å Structure model

(including water structure ) is completely correct & with high accuracy

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Case study 1JSQ, 1PF4, 1Z2R for MsbA and 1S7B,2F2M for EmrE) of (ABC) transporters

102HYD- SAV1866 1Z2R 1Z2R_2HYD_SUPERIMPOSED

PDB Database

(a) Cα-trace, correct structure of the SarA protein (2FRH). (b) Cα-trace, an incorrect model (1FZN).

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Lessons for modellersThe entire protein structure is modelled incorrectly are very

rare. This usually occurs only if the resolution is low and if sensible procedures for model building, refinement and validation are dumped.

every crystal structure, even at atomic resolution, can have problematic parts or aspects Inspection of electron density together with the model may help in identifying such parts(binding site, catalytic residues, interaction, etc.).

From 1 February 2008, good practice in the validation of protein structures: http://xray.bmc.uu.se/embo2001/modval/. 12

2. The structure of the ligand & it’s interactions with the protein are correct

Interactions of Ligand-Receptor are known, understood & correct 13

(a)conformation of the activated (ligand-bound) state of the protein (2GWX).

(b) Identification of a bound fatty acid ligand (cis-vaccenic acid) (2BAW).14

Case study

Difficulties in determining the orientation of asparagine, glutamine and histidine sidechains & assignment of density features to water molecules.

The density for a ligand is poor and the placement of the ligand by the crystallographer is questionable?

modellers can make an important contribution themselves to the structure determination of protein–ligand complexes.

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Lessons for modellers

3. The protein-ligand structure is relevant for drug design

The crystallisation conditions are relevant for drug design

1. pH effect on protein-ligand

SARS coronavirus protease was crystallised at different pH values and in complex with a specific inhibitor.

The structures revealed substantial pH-dependent conformational changes and an unexpected mode of binding for the substrate-analogue inhibitor.

At a pH value of 6 the structure of the monomers in The homodimer differs (one active and other inactive conformation) and the inhibitor binds in a different mode to each monomer.

Case study

2. Protein flexibility

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While an X-ray crystal structure of a ligand bound to its target protein is seductive in its clarity. thermodynamics of the system may confound a simple and straightforward interpretation based on the X-ray crystal model.

Dissimillarity between expected structure–activity relationships and observation from X-ray crystal structures are important.

Combination of X-ray crystal structural information, molecular dynamics simulations and calorimetric investigations is starting to unravel these complexities.

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Can X-ray crystal structures really aid drug design? Used in diseases like cancer, HIV, glaucoma and hypertension. Eg. Aliskiren

To invest in the collection of protein structural information to aid drug design.

potency & selectivity

Many other properties need to be built into the chemical structure that are not directly aided and may even be hindered, by the availability of protein structure models.

Drug design requires careful control of these properties, while maintaining high target affinity.

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Conclusions To prove valuable , provide a strong stimulus to chemical

creativity, through the direct visualisation of the ligand–receptor interactions.

For users of X-ray crystal structure information, however, it is important to realise a crystal structure is a model, a crystallographer’s partly subjective interpretation of experimental data.

This interpretation may be flawed, ambiguous or inaccurate in its details.

To examine the model alongside the experimental electron density and

to put the model to the test through iterations of structure–activity work.

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www.drugdiscoverytoday.com

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