Computer aided drug design

Introduction
Medicinal chemists today are facing a serious challenge because of the increased cost and enormous amount of time taken to discover a new drug, and also because of fierce competition amongst different drug companies

Introduction cont.
It needs approximately 300 to 350 million US $ and 12-13 years for a drug to reach the market http://www/lilly/company/about /highlights/html The hope lies in the rational design of new therapeutic agents

Evolutionary drug designing

Ancient times: products with biological activities used as drugs, exploratory search for novel compounds Chemical Era: organic compounds, natural products, synthetic compounds Rationalizing design process Biochemical era: To elucidate biochemical pathways and macromolecualr structures

Conventional drug design

Conventional strategy for the discovery of new drugs is to take a lead structure and develop analog molecule to exhibit desired Biological properties. The lead is generally found by chance or Random screening.

Old Strategies for drug design and Development

The process involves trial and error approach and is laborious. However, many good drugs are found today using this approach The traditional processes are now supplemented by more direct approaches and are derived from the molecular processes involved in the understanding of the diseases

First generation Rational approach in Drug design

In 1970s the medicinal chemists considered molecules as topological entities in 2 dimension (2D) with associated chemical properties. QSAR concept became quite popular. It was implemented in computers and constituted first generation rational approach to drug design

Old QSAR
The principle of QSAR was later promoted by Hansch and his group (Fujita, 1990). It was based on determination of mathematical equation based on molecular parameters as log (P) (partition coefficient), Steric bulkiness (Es), molecular refractivity(MR) etc. The concept was expanded by quantum chemists who introduced MOMO, LEMO, dipole moments, hybridization moments, molecular polarizability, electronegativity etc

Old QSAR cont

These methods dominated QSAR in 1980s have been very useful to improvise a ligand within the same series . Each of these parameters had been conceptually very relevant. In fact increasing interest was elicited in molecular modeling was a direct consequence of success and limitations of 2D QSAR

Rational Drug design

Scientific principles underlying were known from a long time (E.Fisher 1894, P.Ehrlich 1909) (Lock & Key hypothesis). It was only in seventies that mechanisms underlying were understood. The field has grown after 1980s due to contributions from basic and computational scientists.

2nd generation rational drug design

The acceptance by medicinal chemists of molecular modeling was favored by the fact that the QSAR was now supplemented by 3D visualization. The lock and key complementarity is actually supported by 3D model. Computer aided molecular design (CAMD) is expected to contribute to intelligent lead

 HTS data analysis and binary QSAR, ADME based classification Combinatorial library design Molecular descriptors Compound Clustering Diverse subsets Recursive partitioning

High throughput traditional methods

Objective
1.Introduction to rational drug design and molecular modeling 2.Theoretical methods 3.Analog based drug design 4.Target structure based drug design

1.Introduction to rational drug design

 The concepts of 3-D design are simple. The new molecules are conceived either on the basis of similarity with known reference structure or complementarity with 3-D structure of a known target. The targets could be: Enzyme, Receptors (circulating messenger), Transport systems (ion channels), cell replication and protein synthesis(DNA, RNA) and storage sites

What are the basic concept ?

Ligand Receptor recognition

Sterric complimentarity is necessary but not sufficient. It is a second order effect and not the dominant. 3D database searching is more appropriate than de-novo designing because our knowledge of receptor ligand recognition is poor. De novo designing is good to modify the existing structure to yield more active analog.

1.Shape similarity/ complimentarity

Two homologous molecules may be similar (estrogen and glucocorticoid receptors) or dissimilar (glucocorticoid receptor and GAL-4 protein).

1.Shape similarity/ complementarity cont.

For rotation along jth bond, one can estimate a distance matrix j C =7 Kn n rh
n=1

3D-QSAR programs can estimate shape similarity

1.Problems in shape similarity index

Identity in atomic position is easy to observe but not necessarily true. Dissimilar molecules also may look similar. Two homologous molecules may be similar or totally dissimilar.

Ligand binding site

Ligand binding site can also accommodate significant degree of dissimilarity in ligand structure (eg. - HIV-I protease. Binding site has 10 binding pockets and the enzyme cleaves eight different peptide sequences, with wide structural variation. A slight modification in one sequence can produce effective inhibitor)

2. Electrostatic complementarity
Initial step in formation of a complex molecule eg drug-receptor complex is 'recognition' which must occur at a large distance. Complementarity of charge distribution is an important factor in recognition.

2.Electrostatic complementarity
The 3 D electrostatic field around a molecule (MEP) controls long range interaction between the ligand and the target.

2.Molecular electrostatic potential

MEP depends on : dipole-dipole, dipoleinduced dipole (inductive) and induced dipole-induced dipole (dispersive) interaction between two molecules. QCPE offers number of programs ( Huckel-4, Del Re W Extended Huckel, CNDO, INDO, MINDO, PCILO, ab initio etc) at a reasonable price. There are several public domain programs as:GAMESS, MOPAC etc or from vendors as: (SPARTAN, MOLCAD, DelPhi, HAMOG, DTMM etc).

2.Molecular electrostatic potential

The three dimentional isopotential (MEP) maps can be calculated from electronic distribution around a positively charged nucleus. Latter can be obtained using either X-ray diffraction or quantum chemical methods. In the simplistic approximation one can use point charges on each atom. In a better approximation MEP can also use electronic wave functions.

Visualization of molecular electrostatic map nifedipine by ab initio method 6-31 G** basic set
Contours from -50 kcal/mol (red) to -90 kcal/mol (green) Heiden et al (1993)

MEP as Isopotential plot

Potential calculated using Electrostatic potential by ab initio Gaussian 6-31 G** region from -5 (blue) to 5 kal/mol(red ) calculations on SPARTAN (3.0) Wave function Inc Irvine California USA

Electrostatic potential on Connolly surface for nifedipine using dot surface and solid Triangular surface

Blue negative, Red positive graphics MOLCAD

3.-Hydrophobic complimentarity
In the case of an ampiphilic ligand, in aqueous environment hydrophobic regions try to align themselves. This gives an additional driving force for drug receptor interaction. The solvent partition coefficient P can be used to calculate log( P) as an hydrophobic index by transferring experimental solvent partition coefficient data into hydrophobic fragment constant.

3.Hydrophobic complimentarity
hydrophobic index. n Jj =-2.3 kT 7 fi e -r ij i n - number of fragments, fi =fragment hydrophobicity, Ttemperature Program HINT uses analogy with electrostatic field

Hydropathic field map of nifedipine using HINT

(Kellog et al 1991)
Contours are -8 (red) 4(green)

Molecular lipophilic potential on nifedipine displayed on the Connolly surface

Brown are more lipophilic parts Blue hydrophilic parts Graphics MOLCAD Package SYBYL Tripos St. Louis, Missouri, USA

4.H-bonding complimentarity
H-bonds are localized, depend upon position of 'donors' and 'acceptors' & occur between two strongly electronegative atoms as: O-H..N, O-H..O, N-H..O Recently, C-HO hydrogen bonds have been noticed from crystallographic data. Hydrogen bonding probability maps can be computed on the line of MEP maps.

4.H-bonding complimentarity
Significant amount of tolerance in H-bonds & different ligands makes the problem difficult

4.H-bonding complimentarity
As a result it is thus possible to cocrystallize different ligands in the same protein and yet make good complementarity.

5. Grid fields
At each point, interaction energy between a probe and target molecule is calculated using energy function: Etot = Evdw + Eel + Ehb This can be used to identify points with similar fields and fit them with activity by a 'field fit' method. The program GRID or CoMFA can be used for the same. However to use CoMFA biological activity has to be correctly determined.

Visualization of the molecular interaction field of nifedipine

Favourable Hbonding region from GRID (Goodford et al 1985) using hydrophobic probe (contour level:- -3.5 kcal/ mole) obtained using methyl probe.

Visualization of the molecular interaction field of nifedipine

Contour at -1.4 kal/mol The hydrophobic field represents the location of the hydrophobic amino acid. This can be used for receptor mapping.

Small molecular modeling: Serotonin Receptor Ligands

Molecular interaction field for 5HT2a antagonist FITIT method. 28 Substances (4(phenyketo group, tricyclic, irinadole and buttyrophenone )with known biological activity used (Holtjehydrophobic, green-electron deficient, red Violet- et al electronegative, pink planer ring, pink- protonated N (1995)

Other factors affecting similarity

y There are always numerous superpositions which allow close similarity. How to express similarity- with a definite molecule, with all or with a average picture? y If the binding face of the molecule is unknown, then to what extent should the nonbonding face to be included in determining similarity?

6-Dynamic purturbations in ligand & receptor structures

Conformational flexibility of the target and the drug is important. The bound conformation of the drug need not be the minimum energy conformation.

7-Effect of water molecules at the active site

Important role played by the trapped water molecules at the active site should be considered.

8-Multiple binding sites for the ligand

Most difficult part is possibility of different binding sites. 'Envelope' - or 'active site' has to be defined before applying any technique.

9-Conformational and rotational freedom for the ligands

Very few programs take into consideration the full rotational & translational freedom of the ligand.

No

3-D structure of protein Yes

No

Combinatorial chemistry QSAR, Diverse libraries

Denovo design, Library search Builder,lead discovery

3-D Struct Ligand

Yes

2-D and 3-D QSAR Pharmacophor search Active Analog based Drug Design

Docking, Free energy calculation Ligand optimization

Molecular Modeling Analysis

Small molecular modelling Modeling sets of small molecules Indirect drug design Macromolecular modeling Ligand receptor fit analysis Direct drug design

Design of molecules conforming to the desired requirements

Different approaches to drug design

Direct drug design based on the three dimentional features of the receptors Indirect drug design: analysis based on comparison of three dimentional streochemical and physicochemical features Database search using cheminformatics Three dimentional automated drug design using growers and builders Molecular mimiccry

Rational drug design

Analog based Target structure based

Target based drug designing

Three dimentional structure of macromolecular targets

X-ray Crystallography
Main issues: crystallization/ data collection and analysis improving

NMR spectroscopy
Size of protein and solubility Cost of isotopic labeling

As on 11th Jan 2002 there are 17,300 entries in protein data bank www.rcsb.org

From protein sequence to three-dimensional structure

GENE CHIPS
Readme gene book 1.5 % Expressed genes Probe match/mismatch Affimatrix 20x28 Qm 420,000 probes RIKEN Japan Chips Sequence data base Functional Variability Discovery/Expression for Cancer
Screen mapping

Protein structure modeling

Transforming gene sequence to 3D structure of proteins Rapid development of already known targets New targets related to already known proteins Give structural information to guide drug design

Identification of modeling template

Comparative protein modeling requires at least one sequence of known 3D-structure with significant similarity to the target sequence. In order to determine if a modeling request can be carried out, one compares the target sequence with a database of sequences derived from the Brookhaven Protein Data Bank (PDB), using programs such as FASTA and BLAST.

Template search
Each residue of the reference structure is then aligned with a residue from every other available template structure if their CEatoms are located within 3.0 . This generates a structurally corrected multiple sequence alignment

Aligning the target sequence with the template sequence

The target sequence now needs to be aligned with the template sequence or, if several templates were selected, with the structurally corrected multiple sequence alignment. This can be achieved by using the best-scoring diagonals obtained by SIM.

Framework construction

Loop generation
Using a "spare part" algorithm, one searches for fragments which could be accommodated onto the framework among the Brookhaven Protein Data Bank (PDB) entries determined with a resolution better than 2.5 . Each loop is defined by its length and its "stems", namely the alpha carbon (CE atom co-ordinates of the four residues preceding and following the loop

Non conserved loop generation

The accepted "spare parts" are sorted according to their rmsd, and a CE framework based on the five best fragmentscan be added to the model. In order to ensure that the best possible fragments are used for loop rebuilding, the rmsd cut-off can be incremented from 0.2 onwards until all loops are rebuilt.

Loop generation

Complete backbone generation

Since the loop building only adds CE atoms, the backbone carbonyl and nitrogens must be completed in these regions. This step can be performed by using a library of pentapeptide backbone fragments derived from the PDB entries determined with a resolution better than 2.0 .

Complete backbone generation

These fragments are then fitted to overlapping runs of five Ca atoms of the target model. The co-ordinates of each central tripeptide are then averaged for each target backbone atom (N, C, O) and added to the model. This process yields modelled backbones that differ from experimental co-ordinates by approx. 0.2 rms.

Complete backbone generation

Side chain conformation

Model refinement
Idealisation of bond geometry and removal of unfavourable non-bonded contacts can be performed by energy minimisation with force fields such as CHARMM, AMBER or GROMOS. The refinement of a primary model should be performed by no more than 100 steps of steepest descent, followed by 200-300 steps of conjugate gradient energy minimization

III

Difficulties in docking a Flexible ligand

This method can be used extensively in the conformational search of small molecules.
10 rotatable bonds, it would generate 62 billion conformations If each minimization consumes 1sec, then the complete calculation would require almost 2000 years

Ligand protein interaction

Protein-ligand interaction involve physical contact between the ligand and the receptor for a certain period of time. The receptor-ligand interaction are in general dependent on the concentration of the ligand/protein and salt concentration k+1 RL R+L k-1

Equillibrium constant
Kd=[R][L]/[RL] (G= (,8( S (G change in free energy of reaction, (,=change in enthalpy, (S=change in entropy, T-Temperature, (Go-is lthe free energy change at NTP At equillibrium (G=0, G=kT log Kd/ Co Co Ratio of activities of each chemical species in standard state

Difficulties in comparing theoretically calculated binding energies with Kd

Measurement of Kd., (H and entropy S are not that accurate (Ajay & Murcko 1995). b).Measurement of IC50values: The IC50 values (concentration of the inhibitor required to reduce to binding of a ligand by half) depends on amount of ligand available to the receptor and system (enzyme, cell etc.

Thermodynamics of binidng
The quantity of interest is:(G = Gc - Gu Gc The complexed value of G and Gu the uncomplexed value, Usually we partition (G into various factors Overall loss in entropy due to association i.e. the loss in translational and rotational entropy

Molecular Modelling: Rational Drug Design

Universally used in Lead Generation and Optimisation Structure-Based Design 3D- Structure of the Receptor Docking Analogue-Based Design Bio-activity of a number analogues - QSAR

Topoisomerase-I

Current status
If one has access to sufficient quantity of receptor one can attempt 3D structure by X-ray and NMR then use sophisticated computational approach as thermodynamic cycle perturbation (Kollman and Merz 1990). Angiotesin converting enzyme (ACE) inhibitor was tried extensively without much success

Novel structure
Technology is more robust. Example CAVEAT by Barletts group DOCK by Kuntz

Examples of successful application of CAD

Thrombin inhibitor Ro 46-6240 , BG 8967 in trial Roche Neuroamindase inhibitor 4Guanidino Neu5 Ac2en IGG167 Glaxo Putrine nucleotide phosphate inhibitor Biocryst Pharmaceutical BCX34 Thymidilate Synthatase inhibitor Agouron Carbonic unhydrase inhibitor Trusopt Merck Research

Designed drugs: Enzyme inhibitors

Pargyline Monoamine oxidase Antihypertensine Catropil Antihypertensive Ace Inhibitor Carbidopa Parkinsons disease DOPA decarboxylase Clavulanic acid Antibiotic Fi lactamase Methotrexate Antitumour Dehydrofolate reductase Cytarabine Antileukaemia DNA polymerase Lovastatin Cholesterol lavel HMG CoA reductase

Recepto simulants or blockers

Atracurium Neuromuscular blockade Acetylcholine nicotenic receptor Pirenzepine Antiulcer Muscarine M receptor Fenoldopam Congestive Heart failure Dopamine Dt receptor Butaclamol Antipsychotic Dopamine D2 Prazosine Antihypertensive Adrenergic a receptor Labetalol Antihypertensive Adrenergic a fi recept. Propranolol Antihypertensive adrenergic 13 receptor

Cont
Xamoterol Heart failure adrenergic 13 receptor Salbutamol Antiasthama Simulates adrenergic 132 receptor Ondansetron Antiemetic Serotonin SHT3 receptor Sumatriptan Antimigraine Serotonin SHT1 like receptor Mepyramine Antiallergy Histamine H1 receptors Cimetidine Antiulcer Histamine H2 receptor

Structure Based Drug design

Structure preparation Visualization using graphics tools Active site detection Contact potential Ligand docking

III

Active site finders

Density difference method Alpha shapes Fitting spheres or rectangles etc Each method gives scoring on the basis of number of amino acid side chains in the active cavity Use of genetic modification data is useful for active site finding

Different approaches for docking

The simplest and most popular method is using interactive graphics. At the other end of complexity is full free energy perturbation (FFP) using molecular dynamics approach. In between these two extreme approaches range number of methods which can dock accurately a single ligand or large number of ligands using automated procedures

II

Analog based drug Designing

Pharmacophore search Quantum chemical methods Use of Quantum chemical indices : HOMO and LEMO, Dipole moments etc Molecular electrostatic potential mapping Molecular field analysis Receptor surface mapping Electrostatic surface similarity

Structural similarity and superimposition technique

Horn and Snyder (1971) was the first to using for neuroleptic action of dopamine and chlorpromazine Superimpositions are obvious from 3D structures but not chemical formulae This led to concept of pharmachophore search

Small molecular modeling: Serotonin Receptor Ligands

Molecular interaction field for 5HT2a antagonist FITIT method. 28 Substances (4(phenyketo group, tricyclic, irinadole and buttyrophenone )with known biological hydrophobic, green-electron deficient, red Violet- activity used (Holtje et alpink planer ring, pink- protonated N electronegative, (1995)

2.Molecular electrostatic potential

MEP depends on : dipole-dipole, dipoleinduced dipole (inductive) and induced dipole-induced dipole (dispersive) interaction between two molecules. QCPE offers number of programs ( Huckel-4, Del Re W Extended Huckel, CNDO, INDO, MINDO, PCILO, ab initio etc) at a reasonable price. There are several public domain programs as:GAMESS, MOPAC etc or from vendors as: (SPARTAN, MOLCAD, DelPhi, HAMOG, DTMM etc).

Visualization of molecular electrostatic map nifedipine by ab initio method 6-31 G** basic set
Contours from -50 kcal/mol (red) to -90 kcal/mol (green) Heiden et al (1993)

Electrostatic potential on Connolly surface for nifedipine using dot surface and solid Triangular surface

Blue negative, Red positive graphics MOLCAD

Hydropathic field map of nifedipine using HINT

(Kellog et al 1991)
Contours are -8 (red) 4(green)

Molecular lipophilic potential on nifedipine displayed on the Connolly surface

Brown are more lipophilic parts Blue hydrophilic parts Graphics MOLCAD Package SYBYL Tripos St. Louis, Missouri, USA

Visualization of the molecular interaction field of nifedipine

Favourable Hbonding region from GRID (Goodford et al 1985) using hydrophobic probe (contour level:- -3.5 kcal/ mole) obtained using methyl probe.

Molecular Field Analysis

Activity = 0.947055 - 0.258821(Ele/401) + 0.085612(vdW/392) + 0.122799(Ele/391) - 0.7848(vdW/350)

Comparative Molecular Field Analysis

Electrostatic Field

Steric Field

Blue (electropositive) Red (electronegative)

Green (favourable) Yellow (repulsive) regions

COMFA studies on oxazolone derivatives

q2 = 0.688 and r2 = 0.969 alignment

Steric

Electrostatic

COMSIA studies on imidazole derivatives

alignment Electrostatic

steric Hydrophobic/ hydrophilic

q2 =0.761 and r2 = 0.945

Receptor Surface Analysis

Electrostatic nature of the receptor surface

Purple (Favourable) Green(Unfavourable) drug-receptor surface interactions

Activiy = 0.546816 - 624.921(vdW/1726)2 + 31.65(vdW/505)2 + 602.116(vdW/1563)2 50.1242(-0.1561-wdW/517)2 - 0.11822(Ele/1673)