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Introduction to Protein-Ligand Binding

Authors

Claude Cohen (Synergix), Elie Cohen (Synergix)

Info

The analytical process and principles underlying a structure-based drug design approach are presented and discussed. Methods for analyzing binding are presented and discussed.

Number of Pages: 76 (±1 hours read)

Last Modified: January 2004

Prerequisites: Principles of Rational Drug Design

Introduction

Receptor-Based Drug Design

The aim of receptor structure-based drug design is to create new molecules with high affinity and with macromolecular receptors whose 3D structure is known. The molecules are conceived based on their 3D complementary features with the macromolecular target. In addition, one can exploit the molecular interactions to strengthen the binding of the current prototype (inhibitor, agonist or antagonist) into the active site of the receptor.

Macromolecular Targets

Macromolecular targets are selected on the basis of their role in the biological system involved for the designated disease. Specific biological pathways can either be blocked or magnified to facilitate the desired therapeutic action. The most common targets of drugs are proteins.

Mechanism of Action of Drugs

Drugs act as inhibitors of enzymatic proteins by inhibiting the generation or degradation of molecular components of the cell (e.g. inhibition of enzymes which are essential for bacterial survival by antibiotics). They can act either as receptor agonists by mimicking the effect of an endogenous chemical signal (e.g. nicotine agonists in the treatment of Parkinson disease) or as antagonists, which blocks the signal (e.g. Substance P antagonists for the release of pain).

Drug Targets

Drugs act on antibodies, structural proteins and ion channels (e.g. local anesthetics). Nucleic acids are also frequently used as targets. The interaction can be either direct (e.g. intercalation agents in the treatment of cancer) or indirect through proteins which regulate the replication and expression of genes. Lipids and saccharides also act as drug targets.

Contribution of Recombinant Technologies

The ability to reproduce significant recombinant proteins has had a great impact on the screening process and has facilitated the structure determination of many of these biologically relevant targets.

Operational Strategy: Docking

Docking is an energy-based operation for exploring the binding modes of two interacting molecules. A docking procedure is used as a guide to identify the preferred orientation of a ligand interacting with a macromolecule. The ligand can accommodate small conformational changes to avoid steric repulsions and produce favorable interactions with the receptor.

Analytical Process

The Analytical Process

Receptor-based drug design proceeds in three steps: gathering all relevant data (data collection), in-depth analyses of the information (analysis) and the design of new molecules as a result of the analysis (design).

Data Collection: X-Ray Crystallography

The most current method to determine the 3D structure of macromolecules is by using X-ray crystallography. This technique requires the isolation, purification and crystallization of the protein, which is not always feasible. The results are usually deposited in the Brookhaven Protein Databank, which has more than 20 thousands structures.

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High-Throughput Crystallography for Lead Discovery in Drug Design Blundell TL, Jhoti H and Abell C Nature Rev. Drug Discovery 1 2002

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Protein Structure Prediction Al-Lazikani B, Jung J, Xiang Z and Honig B Curr. Opin. Chem. Biol. 5 2001

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Modelling by Homology Swindells MB and Thornton JM Curr. Opin. Struct. Biol. 1 1991

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Kenakin TP Comprehensive Medicinal Chemistry. Volume 1 Pergamon 1990


Drenth J Principles of Protein X-ray Crystallography Springer Verlag Publishinig 1999


McPherson A Chem. Struct. Approaches Ration. Drug Des CRC Press 1995


McRee DE Practical Protein Crystallography Academic Press 1990


Murray-Rust J Peptide Pharm. Ward Elsevier 1991


Rhodes G Crystallography Made Crystal Clear: A Guide for User's of Macromolecular Models Academic Press 1999

Data Collection: NMR Spectroscopy

NMR techniques and electron diffraction have recently been used but are limited to small proteins (up to 100-120 amino acids).

Data Collection: Homology Models

In the absence of a detailed 3D experimental structure, models of the macromolecular target may be used as surrogates. For example, if the primary sequence of a certain protein is known and it shares a certain degree of sequence homology with one or more proteins for which detailed structural information is available, it is possible to construct a model of the target protein using homology.

Analysis

Now that the raw data has been collected, one must generate additional data that can be deduced from the already existing information. This allows for the proper development of in-depth analyses that will generate new knowledge or hypotheses and the establishment of a rational link between the different components of the information. The aim of the analysis is to create a framework that integrates the knowledge that can be deduced from the structural elements that are essential for the biological activities and the stereo-chemical features of the receptor site.

articles

Molecular Recognition of Protein-Ligand Complexes: Applications to Drug Design Babine RE and Bender SL Chem. Rev. 97 1997

What Can We Learn from Molecular Recognition in Protein-Ligand Complexes for the Design of New Drugs? Bohm H-J and Klebe G Angew. Chem. Int. Ed. Engl. 35 1996

Hydrogen Bonding, Hydrophobic Interactions, and Failure of the Rigid Receptor Hypothesis Davis AM and Teague SJ Angew. Chem. Int. Ed. 38 1999

Recent Developments in Structure-Based Drug Design Klebe G J. Mol. Med. 78 2000

The Maximal Affinity of Ligands Kuntz ID, Chen K, Sharp KA and Kollman PA Proc. Natl. Acad. Sci. U.S.A. 96 1999

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Koehler KF, Rao SN and Snyder JP Guidebook on Molecular Modeling in Drug Design Academic Press 1996

Design Phase

The design process can be initiated when sufficient rationales are established for the creation of molecules conforming to the desired requirements. It aims at the creation of a new molecular entity with optimal binding to the receptor site.

articles

What Can We Learn from Molecular Recognition in Protein-Ligand Complexes for the Design of New Drugs? Bohm H-J and Klebe G Angew. Chem. Int. Ed. Engl. 35 1996

Recent Developments in Structure-Based Drug Design Klebe G J. Mol. Med. 78 2000

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Koehler KF, Rao SN and Snyder JP Guidebook on Molecular Modeling in Drug Design Academic Press 1996

Principles of Analysis

Analysis of the Morphology of the Active Site

The shape and features of the active site are carefully analyzed. Its morphology is thoroughly inspected and broken into components such as pockets and clefts. The analysis of the morphology of an active site is greatly facilitated when a complex between interacting molecules is available.

Morphology of the Active Site of a Protein Kinase

For example the catalytic site of the protein kinases can be well documented based on the binding of ATP, the common cofactor of all these enzymes. One could distinguish the adenine anchorage, the sugar and the phosphate pockets, as well as region I and II whose nature depends on the kinase considered. In the example shown here they both correspond to hydrophobic pockets.

Complexes with Ligands

For data consisting of a complex of the macromolecule with a ligand, the detailed interactions of the ligand are analyzed for good fits, conformational changes, and potential bump areas.

Forces That Contribute to the Binding

Molecular interactions are controlled by different driving forces. The principal forces involved upon the binding of a ligand to a biological system are hydrophobic interactions, hydrogen bonding and electrostatic forces. It has been proposed that the hydrophobic component (which is non-directional) contributes primarily to affinity whereas hydrogen bonding and electrostatic interactions, because of their highly directional nature, contribute principally to specificity.

The Molecular Recognition Process

The main forces involved in molecular recognition are the following: electrostatic, hydrogen bonding and hydrophobic.

Electrostatic

At long-distances, the electrostatic forces are dominant and control the preliminary approach of the ligand molecule to the active site.

Hydrogen Bonding

At medium-range distances, in addition to the electrostatic forces, hydrogen bonds begin to be formed. They start to establish anchorage points for positioning the ligand more precisely.

Hydrophobic

At shorter distances, and this is the last step of the process, hydrophobic forces become the dominant driving force.

Hydrophobic Interactions

In aqueous media hydrophobic groups tend to cluster in order to reduce non-polar surface areas exposed to water. The following table gives a list of the 9 hydrophobic residues out of the 20 naturally occurring amino acid residues present in protein structures.

articles

Hydrophobic Effects. Opinion and Facts Blokzijl W and Engberts JBFN Angew. Chem. Int. Ed. Engl. 32 1993

X-ray Structures of Small Ligand-FKBP Complexes Provide an Estimate for Hydrophobic Interaction Energies Burkhard P, Taylor P and Walkinshaw MD J. Mol. Biol. 295 2000

Aromatic-Aromatic Interaction: a Mechanism of Protein Structure Stabilization Burley SK and Petsko GA Science 229 1985

Hydrophobic Bonding and Accessible Surface Area in Proteins Chothia C Nature 248 1974

3D Molecular Lipophilicity Potential Profiles: A New Tool in Molecular Modeling Furet P, Sele A and Cohen NC J. Mol. Graph 6 1988

CH/pi Interaction in the Packing of the Adenine Ring in Protein Structures Chakrabarti P and Samanta U J. Mol. Biol. 251 1995

Consider Hydrophobic Interactions

The driving force of hydrophobic interactions between a ligand and a receptor is to reduce the exposure of hydrophobic moieties of the ligand to water. In addition, there is a favorable interaction between lipophilic groups in contact (mainly dispersion interactions). High occupancy of hydrophobic pockets of the receptor by a ligand leads to favorable van der Waals attractions, which results in increasing the binding affinity. Therefore, the more empty space in the binding site, the weaker the affinity of the ligand. Note that, although the hydrophobic interactions are weak, there are a lot of them, so the effect can be substantial.

articles

Hydrophobic Effects. Opinion and Facts Blokzijl W and Engberts JBFN Angew. Chem. Int. Ed. Engl. 32 1993

X-ray Structures of Small Ligand-FKBP Complexes Provide an Estimate for Hydrophobic Interaction Energies Burkhard P, Taylor P and Walkinshaw MD J. Mol. Biol. 295 2000

Aromatic-Aromatic Interaction: a Mechanism of Protein Structure Stabilization Burley SK and Petsko GA Science 229 1985

Hydrophobic Bonding and Accessible Surface Area in Proteins Chothia C Nature 248 1974

3D Molecular Lipophilicity Potential Profiles: A New Tool in Molecular Modeling Furet P, Sele A and Cohen NC J. Mol. Graph 6 1988

CH/pi Interaction in the Packing of the Adenine Ring in Protein Structures Chakrabarti P and Samanta U J. Mol. Biol. 251 1995

Elementary Hydrophobic Interactions

Examples of hydrophobic interactions found in X-ray complexes between various amino acids and ligands are presented here.

articles

Hydrophobic Effects. Opinion and Facts Blokzijl W and Engberts JBFN Angew. Chem. Int. Ed. Engl. 32 1993

X-ray Structures of Small Ligand-FKBP Complexes Provide an Estimate for Hydrophobic Interaction Energies Burkhard P, Taylor P and Walkinshaw MD J. Mol. Biol. 295 2000

Aromatic-Aromatic Interaction: a Mechanism of Protein Structure Stabilization Burley SK and Petsko GA Science 229 1985

Hydrophobic Bonding and Accessible Surface Area in Proteins Chothia C Nature 248 1974

3D Molecular Lipophilicity Potential Profiles: A New Tool in Molecular Modeling Furet P, Sele A and Cohen NC J. Mol. Graph 6 1988

CH/pi Interaction in the Packing of the Adenine Ring in Protein Structures Chakrabarti P and Samanta U J. Mol. Biol. 251 1995

Example of Hydrophobic Binding

An example that illustrates the importance of hydrophobic interactions is the design of thyroid hormone analogues. The thyroxine-prealbumin complex was used as a model for the interaction of thyroid hormones with the nuclear thyroid hormone receptor. It was observed that thyroxine fills effectively only five of the six existing pockets of prealbumin. In order to improve the hydrophobic contacts with the sixth pocket, the bromo atom was replaced by a fused phenyl ring (the naphthyl derivative). As expected this substitution led to an increase in binding affinity.

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Computer Graphics in Drug Design: Molecular Modeling of Thyroid Hormone-Prealbumin Interactions Blaney JM, Jorgensen EC, Connolly ML, Ferrin TE, Langridge R, Oatley SJ, Burridge JM and Blake CC J. Med. Chem. 25 1982

Strengthening Hydrophobic Interactions

It can be clearly seen that the naphthyl derivative fills the hydrophobic pocket better than the thyroxine-analog.

Hydrogen Bond Features

Hydrogen bonds are directional and contribute to the positioning of a molecular fragment in a precise orientation in the active site of a protein. Multiple hydrogen bonds can "zip" the ligand into place.

articles

Hydrogen Bonding in Globular Proteins Baker EN and Hubbard RE Prog. Biophys. Mol. Biol. 44 1984

The Role of Hydrogen Bonds in Protein Folding and Protein Association Ben-Naim A J. Phys. Chem 95 1991

Enthalpy of Hydrogen Bond Formation in a Protein-ligand Binding Reaction Connelly PR, Aldape RA, Bruzzese FJ, Chambers SP, Fitzgibbon MJ, Fleming MA, Itoh S, Livingston DJ, Navia MA, Thomson JA and Wilson KP Proc. Natl. Acad. Sci. U.S.A. 91 1994

The Hydrogen Bond in Molecular Recognition Fersht AR Trends. Biochem. Sci. 12 1987

Hydrogen Bond Stereochemistry in Protein Structure and Function Ippolito JA, Alexander RS and Christianson DW J. Mol. Biol. 215 1990

Aromatic Rings Act as Hydrogen Bond Acceptors Levitt M and Perutz MF J Mol Biol. 201 1988

The Nature of Intramolecular Hydrogen-Bonded and Non- Hydrogen-Bonded Conformations of Simple Di- and Triamides Novoa J and Whanghbo MH J. Am. Chem. Soc. 20 1991

Analysis of Hydrogen Bonding Strengh in Proteins Using Unnatural Amino Acids Thorson JS, Chapman E and Schultz PG J. Am. Chem. Soc. 117 1995

Proteins Capabilities in Hydrogen Bonding

A protein has many hydrogen bond donor and acceptor groups. In addition to the backbone peptidic moiety that can form hydrogen bonds with the C=O or the N-H, hydrogen bonds can be formed with the side chains of the residues.

articles

Hydrogen Bonding in Globular Proteins Baker EN and Hubbard RE Prog. Biophys. Mol. Biol. 44 1984

The Role of Hydrogen Bonds in Protein Folding and Protein Association Ben-Naim A J. Phys. Chem 95 1991

Enthalpy of Hydrogen Bond Formation in a Protein-ligand Binding Reaction Connelly PR, Aldape RA, Bruzzese FJ, Chambers SP, Fitzgibbon MJ, Fleming MA, Itoh S, Livingston DJ, Navia MA, Thomson JA and Wilson KP Proc. Natl. Acad. Sci. U.S.A. 91 1994

The Hydrogen Bond in Molecular Recognition Fersht AR Trends. Biochem. Sci. 12 1987

Hydrogen Bond Stereochemistry in Protein Structure and Function Ippolito JA, Alexander RS and Christianson DW J. Mol. Biol. 215 1990

Aromatic Rings Act as Hydrogen Bond Acceptors Levitt M and Perutz MF J Mol Biol. 201 1988

The Nature of Intramolecular Hydrogen-Bonded and Non- Hydrogen-Bonded Conformations of Simple Di- and Triamides Novoa J and Whanghbo MH J. Am. Chem. Soc. 20 1991

Analysis of Hydrogen Bonding Strengh in Proteins Using Unnatural Amino Acids Thorson JS, Chapman E and Schultz PG J. Am. Chem. Soc. 117 1995

Consider Hydrogen Bond Formations

Hydrogen bonds formed between ligand and receptor are at the expense of bonds broken between ligand and solvent and between receptor and solvent. As a result of the release of water molecules, the breaking of their hydrogen bonds and the formation of new hydrogen bonds with the ligand occurred.

articles

Hydrogen Bonding in Globular Proteins Baker EN and Hubbard RE Prog. Biophys. Mol. Biol. 44 1984

The Role of Hydrogen Bonds in Protein Folding and Protein Association Ben-Naim A J. Phys. Chem 95 1991

Enthalpy of Hydrogen Bond Formation in a Protein-ligand Binding Reaction Connelly PR, Aldape RA, Bruzzese FJ, Chambers SP, Fitzgibbon MJ, Fleming MA, Itoh S, Livingston DJ, Navia MA, Thomson JA and Wilson KP Proc. Natl. Acad. Sci. U.S.A. 91 1994

The Hydrogen Bond in Molecular Recognition Fersht AR Trends. Biochem. Sci. 12 1987

Hydrogen Bond Stereochemistry in Protein Structure and Function Ippolito JA, Alexander RS and Christianson DW J. Mol. Biol. 215 1990

Aromatic Rings Act as Hydrogen Bond Acceptors Levitt M and Perutz MF J Mol Biol. 201 1988

The Nature of Intramolecular Hydrogen-Bonded and Non- Hydrogen-Bonded Conformations of Simple Di- and Triamides Novoa J and Whanghbo MH J. Am. Chem. Soc. 20 1991

Analysis of Hydrogen Bonding Strengh in Proteins Using Unnatural Amino Acids Thorson JS, Chapman E and Schultz PG J. Am. Chem. Soc. 117 1995

Elementary Hydrogen Bond Interactions

Examples of hydrogen bond interactions found in X-ray complexes between various amino acids and ligands are presented here.

articles

Hydrogen Bonding in Globular Proteins Baker EN and Hubbard RE Prog. Biophys. Mol. Biol. 44 1984

The Role of Hydrogen Bonds in Protein Folding and Protein Association Ben-Naim A J. Phys. Chem 95 1991

Enthalpy of Hydrogen Bond Formation in a Protein-ligand Binding Reaction Connelly PR, Aldape RA, Bruzzese FJ, Chambers SP, Fitzgibbon MJ, Fleming MA, Itoh S, Livingston DJ, Navia MA, Thomson JA and Wilson KP Proc. Natl. Acad. Sci. U.S.A. 91 1994

The Hydrogen Bond in Molecular Recognition Fersht AR Trends. Biochem. Sci. 12 1987

Hydrogen Bond Stereochemistry in Protein Structure and Function Ippolito JA, Alexander RS and Christianson DW J. Mol. Biol. 215 1990

Aromatic Rings Act as Hydrogen Bond Acceptors Levitt M and Perutz MF J Mol Biol. 201 1988

The Nature of Intramolecular Hydrogen-Bonded and Non- Hydrogen-Bonded Conformations of Simple Di- and Triamides Novoa J and Whanghbo MH J. Am. Chem. Soc. 20 1991

Analysis of Hydrogen Bonding Strengh in Proteins Using Unnatural Amino Acids Thorson JS, Chapman E and Schultz PG J. Am. Chem. Soc. 117 1995

Example of the Hydrogen Bond Binding

The binding of guanine analogs to the active site of purine nucleoside phosphorylase is an excellent example of the importance of the formation of hydrogen bonds. In this case the formation of one additional hydrogen bond increased the biological activity 100 times.

Electrostatic Interactions

Electrostatic interactions occur between polar or charged groups. The following residues contribute to the formation of electrostatic (coulombic or salt-bridge) interactions in proteins. Salt bridges are most stabilizing when they are somewhat shielded from solvent.

articles

Electrostatic Complementarity between proteins and ligands Chau PL and Dean PM J. Comput. Aided Mol. Des. 8 1994

Electrostatic Effects in Proteins Matthew JB Annu. Rev. Biophys. Biophys. Chem. 14 1985

Electrostatic Interactions in Macromolecules: Theory and Applications Sharp KA and Honig B Annu. Rev. Biophys. Biophys. Chem. 19 1990

Elementary Electrostatic Interactions

Examples of electrostatic interactions found in X-ray complexes between various amino acids and ligands are presented here.

articles

Electrostatic Complementarity between proteins and ligands Chau PL and Dean PM J. Comput. Aided Mol. Des. 8 1994

Electrostatic Effects in Proteins Matthew JB Annu. Rev. Biophys. Biophys. Chem. 14 1985

Electrostatic Interactions in Macromolecules: Theory and Applications Sharp KA and Honig B Annu. Rev. Biophys. Biophys. Chem. 19 1990

Strength of Electrostatic Interactions

Electrostatic interactions such as salt bridges and charge-assisted hydrogen bonds appear to be significantly stronger than neutral hydrogen bonds (in the range of -12 and -25 kJ/mol). This interaction energy is moderated by protonic solvents.

articles

Electrostatic Complementarity between proteins and ligands Chau PL and Dean PM J. Comput. Aided Mol. Des. 8 1994

Electrostatic Effects in Proteins Matthew JB Annu. Rev. Biophys. Biophys. Chem. 14 1985

Electrostatic Interactions in Macromolecules: Theory and Applications Sharp KA and Honig B Annu. Rev. Biophys. Biophys. Chem. 19 1990

Example of Electrostatic Interactions

Replacement of the hydroxyl group by an amino substituent resulted in a 400 times more potent neuraminidase (NA) inhibitor.

articles

Influenza Neuraminidase Inhibitors Possessing a Novel Hydrophobic Interaction in the Enzyme Active Site: Design, Synthesis, and Structural Analysis of Carboxylic Sialic Acid Analogues with Potent Anti-Influenza Activity. C.U. Kim et al. J.Am.Chem.Soc. 119 1997

Increase of Potency by the Formation of a Salt Bridge

The increase of potency is due to the formation of a salt bridge between the amino group and the carboxylate of Glu-119.

OH Analog Much Less Potent

The OH analog is 400 times less potent.

Example of Tight Interactions

An Example of a Tight Ligand-Receptor Interaction

Biotin is an enzyme cofactor which functions as a carrier of activated carbon dioxide. One of the tightest binding ligand-receptor pairs known is the complex between biotin and streptavidin (Ki = 0.1 pM). This tight binding is a consequence of high degree of shape and electrostatic complementarity between the ligand and the receptor. In addition, as a result of the binding of the ligand to the receptor, five ordered water molecules from the binding pocket of apo streptavidin are released.

The X-ray Structure of the Biotin/Streptavidin

The crystal structure of the complex Biotin/Streptavidin has been resolved and is visualized here with some details.

The Binding Mode of Biotin with Streptavidin (1/4)

The stabilization of the complex is a result of the favorable interactions between a ligand and a receptor.

The Binding Mode of Biotin with Streptavidin (2/4)

2

The structure of this complex reveals a very complimentary steric fit between the ligand and the protein. Most of the accessible solvent surface of biotin is buried within streptavidin. The only exposed solvent group of biotin is the carboxylate.

The Binding Mode of Biotin with Streptavidin (3/4)

In addition to steric complementarity, there exists a very extended hydrogen bond network:The urea carbonyl makes three hydrogen bonds to the side chains of Tyr43, Ser27 and Asn23 with an approximately tetrahedral geometry.The two urea NH's form hydrogen bonds with Ser45 and Asp128 and the sulfur atom with Thr90.

The Binding Mode of Biotin with Streptavidin (4/4)

The side-chain carboxylate of biotin forms a charge-assisted hydrogen bond with Asn49 and a hydrogen bond with Ser88.

Receptor & Ligand Flexibility

Flexibility of the Receptor

A receptor possesses very flexible structural elements. The following is an example of the HIV-1 protease. X-ray crystallographic studies have shown that the tips of the flaps of this enzyme are capable of undergoing a 7 Å movement between the free and the complex enzyme.

articles

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NMR Relaxation Studies of the Role of Conformational Entropy in Protein Stability and Ligand Binding Stone MJ Acc Chem Res. 34 2001

Flexibility of The Ligand

The flexibility of a ligand has direct consequences on the binding capabilities of the molecule and on the potency of the biological action. To understand ligand interactions, the bioactive conformation of the ligand and its probability of existence are of central importance. Making a flexible molecule rigid is a common strategy used to discover potent analogs.

Entropic Effects

From an entropic viewpoint, many aspects of complex formation are unfavorable as they result in the loss of conformational degrees of freedom for both the protein and the ligand. Therefore, highly favorable enthalpic contacts between the protein and the ligand must compensate for this entropic loss. Moreover, the more rigid the ligand is, the less rotational entropy is lost upon binding, giving higher affinity.

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Structure-Based Design of a Cyclophilin-Calcineurin Bridging Ligand Alberg DG and Schreiber SL Science 262 1993

Rationally Designed Dipeptoid Analogues of CCK. A Free-Wilson/Fijita-Ban Analysis of Some α-Methyltryptophan Derivatives as CCK-B Antagonists Higginbottom M, Kneen C and Ratcliffe GS J. Med. Chem. 35 1992

Design, Synthesis and Biological Activity of Novel Rigid Amidino-phenylalanine Derivatives as Inhibitors of Thrombin Mack H, Pfeiffer T, Hornberger W, Bohm HJ and Hoffken HW J. Enzyme. Inhib. 9 1995

Structure-Based Design of an Inhibitor of the Zinc Peptidase Thermolysin Morgan BP, Bartlett PA, Holland DR and Matthews BW J. Am. Chem. Soc. 116 1994

Role of the Solvent

Solvation and Desolvation

The binding of a ligand to a protein is a complex process in which the interacting entities become partially desolvated. This subtle thermodynamically driven chain of events leads to the formation of favorable interactions between the ligand and the protein. For example upon binding, hydrophobic moieties of the ligand enter into hydrophobic pockets of the receptor to minimize the contact with water.

The Role of the Solvent

The aqueous solvent plays a crucial role in determining the conformational preferences and interaction forces of ligands and receptors. In particular, the hydrophobic effect (also called hydrophobic collapse) is caused by the unfavorable interaction of non-polar groups with water molecules, they tend to cluster together to exclude water and minimize the area of interaction with water. The interpretation of this phenomenon is that water-water interactions are the most favorable interactions and in effect, the water squeezes out the hydrophobes so it can interact with itself, leaving the hydrophobes to interact with themselves.

Relay with Water Molecules

A ligand can bind directly to a receptor or indirectly through water molecules that are located between the ligand and the protein, establishing a hydrogen-bond network between them. The following example illustrates the case of a ligand where one water molecule contributes to the binding of the ligand.

Relay with Several Water Molecules

In addition to one water molecule connecting the protein and the ligand (on the right side of the adenine nucleus), two other water molecules are also contributing to the binding of this ligand (on the left side of the view).

Relay with Water Molecule Having Four H-Bonds

This other example illustrates the binding of a peptide-based molecule to the HIV-1 protease through a water molecule. The water molecule accepts two hydrogen bonds and donates another two; the four hydrogen bonds are in a tetrahedral geometry.

Prediction of Binding Modes

Binding Modes Predicted by Analogy

When an X-ray structure of a complex is available for a given ligand, the design is normally conceived with the assumption that the binding mode of the analog will be similar to that of the reference molecule. This has proven to be valid in many cases.

articles

Do structurally similar ligands bind in a similar fashion? Bostrom J, Hogner A, Schmitt S. J Med Chem. 49(23) 2006 10.1021/jm060167o

Inversion of Binding Modes

However experimental evidence indicates that this assumption may not be always valid. The following illustrates the case of molecules that were assumed to be of the same structure but experimental studies revealed unexpected binding modes. In many cases, the "unexpected" observation simply comes from insufficient development of the analyses.

Inverted Binding Mode of Olomoucine

ATP is the natural ligand of the protein kinases. A number of crystallographic studies of ATP and adenine-like analogs revealed the binding mode of the molecules of this family.

articles

Crystal Structure of Cyclin-Dependent Kinase 2 De Bondt HL, Rosenblatt J, Jancarik J, Jones HD, Morgan DO and Kim SH Nature 363 1993

Multiple Modes of Ligand Recognition: Crystal Structures of Cyclin-dependent Protein Kinase 2 in Complex with ATP and Two Inhibitors, Olomoucine and Isopentenyladenine Schulze-Gahmen U, Brandsen J, Jones HD, Morgan DO, Meijer L, Vesely J and Kim SH Proteins 22 1995

Inhibition of Cyclin-dependent Kinases by Purine Analogues Vesely J, Havlicek L, Strnad M, Blow JJ, Donella-Deana A, Pinna L, Letham DS, Kato J, Detivaud L, Leclerc S and Meijer L Eur. J. Biochem. 224 1994

Pdb

PDB entry: 1HCK

X-Ray Structure of ATP Bound to a Protein Kinase

The X-ray structure of the complex between ATP and the protein kinase CDK2 has been solved and confirms its now classical binding mode observed in all the kinases analyzed.

Intuitive 2D Alignment for Olomoucine

Olomoucine, a relatively selective inhibitor of the CDK2 kinase has an adenine-based scaffold that is also present in ATP. It was therefore anticipated that Olomoucine would have a comparable binding mode, corresponding to the following alignment.

Experimental Binding Mode of Olomoucine

The X-ray structure of the complex of Olomoucine with CDK2 reveals an entirely different binding mode and corresponds to an alignment with ATP that was unexpected.

Origin of the Inverted Binding Mode of Olomoucine

Modeling analyses show that for steric reasons the Olomoucine molecule cannot conform to the binding of the authentic ligand ATP. The molecule binds to the catalytic site in a different orientation and another network of hydrogen bonds.

Inverted Binding Mode of Methotrexate

Dihydrofolate and methotrexate bind to the catalytic site of dihydrofolate reductase. Both have a rather long side chain (R) that is the same in the two molecules.

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Dihydrofolate Reductase: X-ray Structure of the Binary Complex with Methotrexate Matthews DA, Alden RA, Bolin JT, Freer ST, Hamlin R, Xuong N, Kraut J, Poe M, Williams M and Hoogsteen K Science 197 1977

Methotrexate Binding to Dihydrofolate Reductase Roberts GC, Feeney J, Birdsall B, Charlton P and Young D Nature 286 1980

Matching Chemistry and Shape in Molecular Docking Shoichet BK and Kuntz ID Protein. Eng. 6 1993

Pdb

PDB entry: 3DFR

PDB entry: 4DFR

PDB entry: 6DFR

Intuitive 2D Alignment for Methotrexate

By simple observation of their chemical structures, it seems logical to imagine that the two molecules adopt similar binding modes, with the two structures aligned in the following way.

Experimental Binding Mode of Methotrexate

The X-ray structures of the complexes between the dihydrofolate and the methotrexate molecules indicate an entirely different binding mode for the two molecules, each molecule binding to the catalytic site upside down, relative to the other, while the methotrexate was found to be protonated.

Origin of the Inverted Binding Mode of Methotrexate

This can be understood by the better match of 3D distribution of the electrostatic properties of the two molecules by inverting one of them.

Binding Mode Predicted from SAR

When a sufficient number of analogs of a lead molecule are synthesized and tested, analysis of the resulting structure-activity relationships allows one to visualize the probable complementary receptor surface of this series of analogs.

Methods for Analyzing Binding

Analyzing Ligand-Receptor Binding

Computer graphics provide a powerful tool for analyzing the fit of a ligand into a complex macromolecular environment. Moreover computerized methods provide quantitative content to the detailed interaction energies involved in the binding of a molecule.

Ligand-Binding Predictions

The accurate and fast prediction of the binding energy for a protein-ligand complex is very important for the effective design and optimization of lead compounds. However, this is not an easy task. The binding of a ligand to a protein is a complex process.

Visual Analyses

Advanced computer graphics systems including stereo, real time rotations, volume and surface rendering provide in-depth analyses of the 3D stereo-chemical features of the active site of a protein and the favorable or unfavorable interaction of atoms or groups of atoms.

Docking Analyses

There are many forces that are involved in the intermolecular association or binding between two molecules. An energy-driven docking procedure that takes into account van der Waals, hydrogen bonding and electrostatic forces allows one to "dock" a ligand to a binding site by optimizing the interaction of the ligand and its receptor.

articles

A Good Ligands is Hard to Find: Automated Docking Methods Blaney JM and Dixon JS Perspect. Drug Discov. Design 1 1993

Docking Flexible Ligands to Macromolecular Receptors by Molecular Shape DesJarlais RL, Sheridan RP, Dixon JS, Kuntz ID and Venkataraghavan R J. Med. Chem. 29 1986

Recent Advances in Structure-based Rational Drug Design Gane PJ and Dean PM Curr. Opin. Struct. Biol. 10 2000

Docking Small-Molecule Ligands into Active Sites Jones G and Willett P Curr. Opin. Biotechnol. 6 1995

Theory of Macromolecule-Ligand Interactions Kollman PA Current Opinion in Structural Biology 4 1994

Geometric Approach to Macromolecule-Ligand Interactions Kuntz ID, Blaney JM, Oatley SJ, Langridge R and Ferrin TE J. Mol. Biol. 161 1982

Structure-based Strategies for Drug Design and Discovery Kuntz ID Science 257 1992

Ligand-Protein Docking and Rational Drug Design Lybrand TP Curr. Opin. Struct. Biol. 5 1995

Evaluation of Docking Functions for Protein-Ligand Docking Perez C and Ortiz AR J. Med. Chem. 44 2001

Flexible Docking and Design Rosenfeld R, Vajda S and DeLisi C Annu. Rev. Biophys. Biomol. Struct. 24 1995

Prediction of the Structure of a Receptor-Protein Complex Using a Binary Docking Method Stoddard BL and Koshland DE Jr Nature 358 1992

A Review of Protein-Small Molecule Docking Methods Taylor RD, Jewsbury PJ and Essex JW J. Comput. Aided. Mol. Des. 16 2002

Docking Ligands onto Binding Site Representations Derived from Proteins Built by Homology Modelling Schafferhans A and Klebe G J. Mol. Biol. 307 2001

Manual Docking with Computer Graphics

In manual methods ligands are constructed interactively in the active site of a macromolecular target by using computer graphic systems and then the complex is minimized. However, it is not easy to find the global-energy-minimum structure because many alternative conformations need to be explored to identify the preferred binding mode of a given molecule.

Automated Methods for Docking

Automated docking methods allow one to explore a number of possibilities in a systematic way. They allow a much more complete exploration of alternative binding modes as compared to interactive manual docking.

Calculation of Binding Energies

The accurate and fast prediction of the binding constant for a protein-ligand complex is very important for the effective design and optimization of lead compounds. Goodford proposed to evaluate the interaction energy on a grid of test points surrounding the target. This exploration reveals "hot areas" where favorable interactions can take place.

articles

Computational Methods to Predict Binding Free Energy in Ligand-Receptor Complexes Ajay MMA and Murcko MA J. Med. Chem. 38 1995

The Effect of Multiple Binding Modes on Empirical Modeling of Ligand Docking to Proteins Brem R and Dill KA Protein Sci. 8 1999

The Statistical-Thermodynamic basis for Computation of Binding Affinities: A Critical Review Gilson MK, Given JA, Bush BL and McCammon JA Biophys. J. 72 1997

A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules Goodford PJ J. Med. Chem. 28 1985

A Priori Prediction of Ligand Affinity by Energy Minimization Holloway MK Perspect. Drug Discov. Des. 9-11 1998

Computer Modeling of the Interactions of Complex Molecules Kollman PA and Merz KM Jr Acc. Chem. Res. 23 1990

Rapid Estimation of Relative Binding Affinities of Enzyme Inhibitors Reddy MR, Viswanadhan VN and Erion MD Perspect. Drug Discov. Des. 85 1998

book

Van Gunsteren WF

ESCOM 1989

Free Energy Perturbation Techniques

Free energy perturbation (FEP) calculation is an approach developed to calculate binding energies. Using a closed thermodynamic cycle (where the free energy between two states is independent of the path considered), molecular dynamics, and the experimental binding free energy of one inhibitor (ΔG1), FEP allows one to calculate free energy of new inhibitors (ΔG3). The method requires substantial computer time and thus it should not be used on a routine basis.

articles

Methodological Advances in Molecular Dynamics Simulations of Biological Systems Brooks CL 3rd Curr. Opin. Struct. Biol. 5 1995

Free Energy Calculations: Application to Chemical and Biochemical Phenomena Kollman P Chem. Rev. 93 1993

Free Energy from Simulations McCammon JA Curr. Opin. Struct. Biol. 1 1991

Free Energy Pertubation Simulation of the Inhibition of Thermolysin: Prediction of the Free Energy of Binding of a New Inhibitor Merz KM and Kollman PA J. Am. Chem. Soc. 111 1989

Absolute and Relative Binding Free Energy Calculations of the Interaction of Biotin and Its Analogs with Streptavidin Using Molecular Dynamics Free Energy Perturbation Approaches Miyamoto S and Kollman PA Proteins 16 1993

A New Method for Carrying Out Free Energy Perturbation Calculations: Dynamically Modified Windows Pearlman DA and Kollman PA J. Chem. Phys. 90 1989

Calculations of Relative Differences in Binding Free Energy of HIV-1 Protease Inhibitors: a Thermodynamic Perturbation Approach Reddy MR, Varney MD, Kalish V, Viswanadhan VN and Appelt K J. Med. Chem. 37 1994

Computer Simulation of Molecular Dynamics: Methodology, Applications and Perspectives in Chemistry Van Gunsteren WF and Berendsen HJC Angew. Chem. Int. Ed. Engl. 29 1990

Dynamics and Design of Enzyme Inhibitors Wong CF and McCammon JA J. Am. Chem. Soc. 108 1986

Energies from Force Field Calculations

The binding constant (K) of a ligand to its receptor is related to the ligand/receptor interaction energy (ΔH) via: ΔG = ΔH - TΔS = -RT lnK. If we assume the entropy change is the same for each molecule then we can write: ΔH = - RT lnK where the entalpy change on binding (interaction energy) is proportional to the log of the binding constant. The interaction energy can be calculated by force-field calculation as the sum of van der Waals and electrostatic interactions between the ligand and the receptor.

articles

Computer Modeling of the Interactions of Complex Molecules Kollman PA and Merz KM Jr Acc. Chem. Res 23 1990

Correlation with Biological Activities

This hypothesis has been shown to work in a handful of systems. In the case of HIV-1 protease inhibitors, we have X-ray data of co-crystallized ligand/receptor complexes of homologous series. A high correlation (r = 0.78) between the calculated interaction energy and biological activity (IC50) of a training set of 33 closely related HIV-1 protease inhibitors was observed. It was employed successfully to predict the potency of potential inhibitors such as the indane derivative, which is a precursor of indinavir.

Energies from Scoring Functions

Scoring functions are algorithms that assign a numerical score to a given ligand inside a receptor. The score should ideally give a measure of the ligand-protein binding constant. "Complementarity Score" are of moderate accuracy; the following illustrates the results obtained in the prediction of the binding constants of thermolysin inhibitors.

articles

The Development of a Simple Empirical Scoring Function to Estimate the Binding Constant for a Protein-Ligand Complex of Known Three-Dimensional Structure Bohm H-J J. Comput. Aided Mol. Design 8 1994

Knowledge-Based Scoring Function to Predict Protein-Ligand Iinteractions Gohlke H, Hendlich M and, Klebe G J. Mol. Biol. 295 2000

Evaluation of Docking/Scoring Approaches: A Comparative Study Based on MMP3 Inhibitors Ha S, Andreani R, Robbins A and Muegge I J Comput. Aided Mol. Des. 14 2000

Scoring Functions: a View from the Bench Tame JRH J. Comput. Aided. Mol. Des. 13 1999

Limitations of Scoring Functions

One of the limitations of scoring functions is that the conformational energy of the ligand is not taken into account. The predictions can be improved by calculating the difference of free energy between the docked conformation and the global minimum of the ligand.

Calculating Desolvation Energies

Accounting for solvent effects (e.g solvation/desolvation energies) is important for understanding of ligand-receptor interactions. Methods are now available to model the effects of solvents on both small and macromolecular solutes ranging from solvent continuum models to the incorporation of discrete solvent shells.

articles

An SCF Solvation Model for the Hydrophobic Effect and Absolute Free Energies of Aqueous Solvation Cramer JC and Truhlar DG Science 256 1992

Solvation Energy in Protein Folding and Binding Eisenberg D and McLachlan AD Nature 319 1986

Molecular Modeling in Solvent Milne GWA, Nicklaus MC and Hodoscek M J. Mol. Struct. 291 1993

Accessible Surface Areas as a Measure of the Thermodynamic Parameters of Hydration of Peptides Ooi T, Oobatake M, Nemethy G and Scheraga HA Proc. Natl. Acad. Sci. U.S.A. 84 1987

Semianalytical Treatment of Solvation for Molecular Mechanics and Dynamics Still WC, Tempczyk A, Hawley RC and Hendrickson T J. Am. Chem. Soc. 112 1990

Surface Area Included in Energy Refinement of Proteins, A Comparative Study on Atomic Solvation Parameters von Freyberg B, Richmond TJ and Braun W J. Mol. Biol. 233 1993

Atomic Solvation Parameters Applied to Molecular Dynamics of Proteins in Solution Wesson L and Eisenberg D Protein Science 1 1992

Empirical Solvation Models in the Context of Conformational Energy Searches: Application to Bovine Pancreatic Trypsin Inhibitor Williams RL, Vila J, Perrot G and Scheraga HA Proteins 14 1992

Inclusion of Solvation in Ligand Binding Free Energy Calculations Using the Generalized-Born Model Zou X, Sun Y and Kuntz ID J. Am. Chem. Soc. 121 1999

Conclusion

Conclusion

To summarize, receptor-based drug design is an approach, ligand-receptor complementary features is a concept, and docking treatments is a technique.

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