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JOURNAL OF MOLECULAR RECOGNITION J. Mol. Recognit. 2002; 15: 405–422 Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/jmr.597

Protein–protein interactions: mechanisms and modification by drugs A. V. Veselovsky1*, Yu. D. Ivanov1, A. S. Ivanov1, A. I. Archakov1, P. Lewi2 and P. Janssen2 1

Institute of Biomedical Chemistry, Moscow, Russia Center for Molecular Design, Janssen Pharmaceutica N.Y., Belgium

2

Protein–protein interactions form the proteinaceous network, which plays a central role in numerous processes in the cell. This review highlights the main structures, properties of contact surfaces, and forces involved in protein–protein interactions. The properties of protein contact surfaces depend on their functions. The characteristics of contact surfaces of short-lived protein complexes share some similarities with the active sites of enzymes. The contact surfaces of permanent complexes resemble domain contacts or the protein core. It is reasonable to consider protein–protein complex formation as a continuation of protein folding. The contact surfaces of the protein complexes have unique structure and properties, so they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations have been undertaken to find or design small molecules that block protein dimerization or protein(peptide)– receptor interaction, or on the other hand, induce protein dimerization. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: protein–protein interaction; protein surface; inhibitors; drug design; antagonist; protein complex; thermodynamics; dimerization Received 8 Junuary 2002; revised 18 March 2002; accepted 28 June 2002

INTRODUCTION Protein–protein interaction is a common mechanism responsible for functioning of numerous processes in the cell. Protein complex formation is crucial for formation of active sites of oligomer enzymes and maintenance of their effective conformation (Banci et al., 1998; Holwerda, 1999). It is also crucial for numerous regulatory processes, including signal transduction (Eyster, 1998; Klemm et al., 1998; Souroujon and Mochly-Rosen, 1998), cell–cell contacts (Alattia et al., 1999), electron transport systems (respiratory chain, cytochrome P450 oxygenase system; Cunha et al., 1999; Schenkman and Jansson, 1999), antigen–antibody interaction (Dall’Acqua et al., 1998; Salzmann and Bachmann, 1998), DNA synthesis (Dear et al., 1997; Sengchanthalangsy et al., 1999), formation of intracellular structures (Herrmann and Aebi, 1998; Hilpert et al., 1999) etc. Pathological protein complex formation may be responsible for the development of some components of Alzheimer’s and prion diseases (Cohen and Prusiner, 1998; Selkoe, 1998). Furthermore, protein aggregation may occur as a result of protein extraction and subsequent protein purification; this is typical, especially for membrane proteins (Kiselyova et al., 1999). In these cases, proteins bind each other rather unspecifically and this leads *Correspondence to: A. V. Veselovsky, Institute of Biomedical Chemistry RAMS, Pogodinskaya str. 10, Moscow 119992, Russia. E-mail: [email protected] Contract/grant sponsor: RFBR; contract/grant number: N 99-04-48081; contract/grant number: N 01-04-48128. Abbreviations used: HIV, human immunodeficiency virus; hGH, human growth hormone; ID, inhibitor of dimerization; PH, pleckstrin homology; SH2, Src-homology-2; SH3, Src-homology-3.

Copyright # 2002 John Wiley & Sons, Ltd.

to artificial generation of protein complexes with irregular structure. Depending on the stability and mechanism of protein– protein complex formation protein complexes can be subdivided into non-obligate (short-living) complexes and permanent stable complexes (proteins are native only in oligomeric structures; Jones and Thornton, 1996, 2000). Tsai et al. (1997b) proposed two-state and three-state models for such complex formation. According to the former model, contacting proteins can ‘exist either unfolded or folded together in a complex’, whereas the three-state model of complexes implies that each protein folds separately and only after that can folded proteins form the complex (Tsai et al., 1997b). So the formation of the permanent complexes can be considered as a continuation of protein folding. It is reasonable to suggest that the structure and properties of the interfaces of the proteins forming these two types of complexes must differ in their structure and properties. Currently, a huge amount of information about various aspects of protein–protein interactions has been accumulated. In this review, we consider the common mechanisms underlying physiological protein–protein interactions and recent achievements in the design of compounds regulating complex formation or disintegrating pre-existing complexes. These studies culminate in the development of a new class of biologically active low-weight non-peptide compounds modifying protein–protein interactions.

STRUCTURE AND PROPERTIES OF PROTEIN–PROTEIN CONTACTS Most information about protein contact areas was obtained

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from analysis of three-dimensional structures solved by X-ray crystallography or NMR. Some useful information was obtained using mutagenic screening (Bogan and Thorn, 1998; Dall’Acqua et al., 1998; Massova and Kollman, 1999), site-directed mutagenesis (Martin et al., 1999b; Ortiz et al., 1999; Otzen and Fersht, 1999; Vaughan et al., 1999) and chemical modification (Fancy and Kodadek, 1999; Ubarretxena-Belandia et al., 1999). Other methods were also employed for analysis of protein–protein contacts. They included fluorescent methods (Bhattacharya et al., 1996; Park and Raines, 1997; Sloan and Hellinga, 1998), calorimetric analysis (Aoki et al., 1998a; Leavitt, Freire, 2001), the two-hybrid system (Hu et al., 2000a), biosensor methods (Glaser and Hausdorf, 1996; Ivanov et al., 1999a,b, 2001; McDonnell, 2001; Rich and Myszka, 2001) etc. (Appling, 1999; Beeckmans, 1999; Ehring, 1999; Kameshita et al., 1998; Rudert et al., 1998; Rudiger et al., 1999; Velev et al., 1998; Vergnon and Chu, 1999; Viani et al., 2000). The shape of the protein interface The contact surface area consists of 6–30% of the monomer ˚ 2. The surface area and may vary from 550 to 4900 A average value of the contact surface of monomers is about ˚ 2 (Jones and Thornton, 1996; Stites, 1997). There is a 800 A poor correlation between solvent accessible surface area of the permanent complex and its molecular weight. No correlation was found for non-obligate complexes, although their interfaces were more planar (Jones and Thornton, 1996). Amino acid composition Repeated attempts were undertaken to determine possible enrichment of amino acid residues in the protein contact interfaces with certain amino acids. Some authors detected an increased number of arginine, histidine, asparagine, tryptophan, tyrosine and serine residues in the contact regions in comparison with their common content in the protein (Stites, 1997). Some authors found increased content of aromatic amino acids (Davies and Cohen, 1996) or hydrophobic amino acid residues (Jones and Thornton, 1996; Ivanov et al., 1999a, 2000). Such a variety of the data may reflect different sets of protein complexes used for the analysis. Another reason for such diversity may be the different nature of the analysed complexes. Since the contact interfaces of permanent complexes are similar to the protein core (Tsai et al., 1996, 1997a), the hydrophobic amino acids apparently predominate (Jones and Thornton, 1996; Stites, 1997). However, in non-obligate complexes, where contact with the aqueous environment is quite possible, hydrophilic and charged amino acids may predominate (Jones and Thornton, 1996; Ivanov et al., 1999b, 2001). So it seems unlikely that the contact interface is actually characterized by an increased proportion of certain amino acids. It is possible that the prevalence of some amino acids in certain contact surfaces reflects the specific properties of protein areas involved in these interactions. Copyright # 2002 John Wiley & Sons, Ltd.

Secondary structure All types of secondary structure (helices, beta-sheets, turns and random coil) have been found in contact areas of the interacting proteins (Stites, 1997; Tsai et al., 1997a). Analysis of 225 complexes revealed the following range of distribution of the secondary structures in interface areas: random coil (47%) >a-helix (36%) >b-sheet (17%). The distribution of the second structures in contact interfaces depends on the type of the complex formed (permanent or non-obligate; Jones and Thornton, 1996). The architecture of the permanent complex interfaces is similar to the protein core, exhibiting limited sets of protein folding patterns. The distribution of secondary structures in the interface of nonobligate complexes shows larger variability and resembles exterior protein surfaces with the exception of a higher ratio of helices (Tsai et al., 1997a). Usually contact area represents short segments of the secondary structures. The protein contact interfaces include from 1 to 15 such segments (Jones and Thornton, 1996). In most cases, the interface surfaces consist of various types of secondary structure, but a single type of secondary structure was also found there (Jones and Thornton, 1996). Frequently various proteins involved in protein complex formation have stable structural domains denominated with their own titles. They include helix–loop–helix domains (Ghosh and Chmielewski, 1998; Norton et al., 1998), SH2 (Src-homology-2) (Pawson, 1995; Pawson et al., 2001), SH3 (Src-homology-3; Pawson, 1995), PH (pleckstrin homology; Pawson, 1995), PDZ (Songyang et al., 1997) and PDZ2 (Kozlov et al., 2000) domains and others (Blatch and Lassle, 1999; Schumacher et al., 2000; Zhang et al., 1998; Fig. 1). Similar stable structural domains in different proteins can participate in the regulation of various cell processes (Ahmad et al., 1998; Zhang et al., 1998). These domains recognize and bind to certain short peptide motifs. For example, the SH3 domain recognizes a proline-rich motif (Pawson, 1995). One protein with such a domain can bind several different proteins (Gotz et al., 1999; Foti et al., 1999; Onofri et al., 2000; Souroujon and Mochly-Rosen, 1998) or one protein can contain several such domains (Dobrosotskaya et al., 1997; Dong et al., 1998). Forces involved in protein–protein interactions Steric, hydrophobic, electrostatic interactions and hydrogen bonds are the main factors responsible for protein–protein interactions. (a) Steric complementarity. Analysis of protein contacts revealed that their interface surfaces are quite complementary to each other (Jones and Thornton, 1996; Stites, 1997; Tsai et al., 1997b). The degree of complementarity depends on the type of protein interaction. The permanent complexes exhibit highest complementarity. Non-obligate complexes and protein–inhibitor complexes are characterized by lower complementarity, and antigen–antibody complexes have the worst complementarity (Jones and Thornton, 1996; Stites, 1997; Tsai et al., 1997b). Usually protein interfaces in protein complexes contain some cavities. Analysis of the interface surfaces of 24 intersubunit contacts showed that J. Mol. Recognit. 2002; 15: 405–422

PROTEIN–PROTEIN INTERACTIONS AND DRUGS

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Figure 1. Some examples of structure of stable domains with their interacting peptides. (A) PDZ domain from neuronal nitric oxide 2 synthase (PDB1B8Q); (B) SH2 domain from phosphotransferase (PDB1SPS); (C) SH3 domain from FYN proto-oncogene 2 tyrosine kinase (PDB1A0N). Interacting peptides are shown as sticks.

only two of them do not have such cavities. Usually cavity surfaces represent about 10% of total interface surfaces. Most cavities (about 63%) are filled with solvent (Hubbard and Argos, 1994). (b) Hydrophobic interaction. The important contribution of the hydrophobic force to the protein–protein interaction has been demonstrated in numerous studies (Eisenhaber and Argos, 1996; Tsai et al., 1996, 1997a; Wells, 1996). The average values of the hydrophobicity of contact surfaces usually represent a mean of the hydrophobicity of the protein core and its surface (Jones and Thornton, 1996; Tsai et al., 1997a). The contribution of the hydrophobic interaction is higher in permanent complexes than in nonobligate complexes (Jones and Thornton, 1996). The latter can be explained by the fact that permanent complexes usually exist in the bound state and the hydrophobic force is more preferential for this purpose, whereas non-obligate complexes are assembled in the water environment for rather a short time, and this makes energetically unfavourable the high hydrophobicity of their surfaces. However the non-obligate complexes of membrane proteins, such as cytochrome P450 2B4, in contrast to water-soluble proteins, are formed by hydrophobic interaction of their membrane parts (Ivanov et al., 1999a, 2000). In the case of enzyme interaction with peptide inhibitors or substrates, the contacted interfaces may have hydrophilic surfaces (Jones and Thornton, 1996; Stevens et al., 2000). The hydrophobic regions in the contact interfaces are organized as patches. The number of such patches may vary ˚ 2, but from 1 to 15. Usually their sizes are within 200–400 A 2 ˚ (Lijnzaad and Argos, 1997). they can achieve 3000 A Analysis of complementarity of the large patches of interfaces from different subunits showed their low overlap of each other (Lijnzaad and Argos, 1997). (c) Electrostatics. The electrostatic force is the other significance force involved in protein–protein interactions (Gong et al., 2000; Grucza et al., 2000; Muegge et al., 1998; Sheinerman et al., 2000; Stevens et al., 2000; Xu et al., 1997a; Zeng et al., 1999; Ivanov et al., 2001). Originally it was assumed that the charges on the contacting surfaces are located complementarily to each other; however, the modern viewpoint suggests the electrostatic complementarity of interacting protein surfaces (McCoy et al., 1997). Copyright # 2002 John Wiley & Sons, Ltd.

The charge density varies from 0 to 12 charged groups per interface surface (Xu et al., 1997b). The distribution of the opposite charges in the interfaces of the contacting area showed that salt bridges across them are highly favourable (Drozdov-Tikhomirov et al., 2001; Xu et al., 1997a,b). The desolvation cost of the charged groups in salt-links is lower, since they have favourable interactions with other charges and hydrophilic residues surrounding them (Xu et al., 1997a). It was proposed that a long-range attractive electrostatic force could promote formation of encounter complexes and therefore accelerate the rate of complex formation (for review see Gabdoulline & Wade, 1999). Also the electrostatic interaction can define the lifetime of complexes (Archakov and Ivanov, 1999). (d) Hydrogen bonding. The average number of hydrogen bonds is proportional to the area of subunit interfaces: one ˚ 2 (Jones and Thornton, 1996) or bond for each 100–200 A about 10 bonds per interface (Lo Conte et al., 1999; Xu et al., 1997b). The hydrogen bonds are preferably of oxygen– nitrogen type (Xu et al., 1997b). The major proportion of the hydrogen bonds is formed by side chains of amino acids (about 76% of all hydrogen bonds). The exceptions are bsheet interfaces as for HIV protease (Wlodawer and Vondrasek, 1998), or protein complexes with peptide inhibitor or substrate (Jones and Thornton, 1996), where the groups of the main chain usually form hydrogen bonds. However, the hydrogen bonds in protein interfaces are usually not in the optimal position, so they ‘are normal or weak in terms of energetics’ (Xu et al., 1997b). Some hydrogen bonds are formed between protein contact surfaces and water molecules located near them (Tsai et al., 1996; Xu et al., 1997b). Contrary to hydrogen bonds formed between protein surfaces, the protein–water hydrogen bonds are ‘good’ ones (Xu et al., 1997b). Since water molecules form more than one hydrogen bond they can interact with a protein group and with another water molecule, forming a network in protein–protein interfaces (Davies and Cohen, 1996; Janin, 1999; Xu et al., 1997b). (e) Water in interfaces. Water molecules are frequently present at the complex interfaces (Davies and Cohen, 1996; Janin, 1999; Wells, 1996). The number of the water molecules usually varies from 1 to 50 (Davies and Cohen, 1996). Water molecules surround the contacting interfaces J. Mol. Recognit. 2002; 15: 405–422

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or are buried in them (Davies and Cohen, 1996; Larsen et al., 1998). In the latter case, they are located in the cavities of the protein interfaces (Dall’Acqua et al., 1998; Vaughan et al., 1999). The water molecules in the cavities may be highly coordinated (Pardanani et al., 1998). They form hydrogen bonds with protein groups and other water molecules, and results this in aqueous networks along the protein interfaces (Dall’Acqua et al., 1998; Janin, 1999; Xu et al., 1997b). Interface water molecules stabilize the protein complexes by forming additional hydrogen bonds, by interacting with charges, and by increasing shape and charge complementarity (Janin, 1999; Larsen et al., 1998; Li et al., 2000; Pardanani et al., 1998; Xu et al., 1997b). Protein–protein binding is accompanied by partial desolvation of the contacted surfaces and this predominates in complexes in which one of reactants is neutral or weakly charged (Camacho et al., 1999, 2000). (f) Conformation. In some cases, considerable differences in the structures of the protein monomers and their complexes were not found (Jones and Thornton, 1996; Muegge et al., 1998). However, most studies revealed various structural changes occurring upon complex formation. These changes were denoted ‘induced-fit’ effects (Betts and Sternberg, 1999; Decrescenzo et al., 2000; Kimura et al., 2001; McCammon, 1998; Sundberg and Mariuzza, 2000). Protein–protein interaction can induce changes in the positions of the side chains of amino acids, motion of the main chain (especially if it is a loop), or domain (Betts and Sternberg, 1999; Carr et al., 1997; Davies and Cohen, 1996; Jones and Thornton, 1996; Wall et al., 1998). The analysis of the conformational changes in lysozyme induced by binding of various antibodies showed ˚ (Davies that some amino acids could deviate by up to 8 A and Cohen, 1996). It was shown that the rearrangement in the protein backbone appeared to be due to low-energy conformational changes, which enable H-bond formation and packing of the amino acid residues (Janin, 2000). The difference in data may be attributed to mechanisms responsible for conformational changes during assemble of permanent and non-obligate complexes. The former operate during protein folding, which is accompanied by mutual optimization of the interacting protein structures. Proteins possessing prefolded structures form non-obligate complexes. They have limited conformational freedom for maximal optimization of the subunit structures. This results in formation of cavities, the presence of water molecules at the complex interfaces, non-optimal hydrogen bond geometry (Dall’Acqua et al., 1998; Vaughan et al., 1999; Xu et al., 1997b) etc. The driving force for protein structure adaptation is the decrease of free energy of the complexes.

Thermodynamics and kinetics of protein–protein interactions Thermodynamics gives the theoretical basis for understanding the processes of protein–protein interaction. The formation of the protein–protein complex may be written as: AB

ka !AB kd


1

where kd is the first-order rate constant for the dissociation reaction and ka is the second-order rate constant for the association reaction. Their ratio is the equilibrium constant for association (Ka) or for dissociation (Kd) according to the law of mass action that is usually written as: AB 1 kd Kd AB Ka ka

2

although the use of the values of activity instead of reactant concentrations is more correct. The thermodynamic parameters have been determined by numerous experimental methods: calorimetric (isothermal titration and differential scanning calorimetry) methods (Aoki et al., 1998a; Leavitt and Freire, 2001), UV–vis absorption methods (Lehnerer et al., 1998), fluorescence methods (Davydov et al., 1996), and analytical ultracentrifugation (Chirlando et al., 1995). Recently optical biosensor methods have been introduced. These methods are of two types, resonant mirror (Ivanov et al., 1999a, 2001) and surface plasmon resonance (Glaser and Hausdorf, 1996). These types allow recording of complex formation in real time (without special labels) by recording the change of refraction index of the medium during the complex formation. The interrelationship between the main thermodynamic parameters characterizing complex formation, such as Gibbs free energy (DG), enthalpy change (DH), entropy change (DS), heat capacity difference (DCp) can be described by the following equations: G0

RT ln Kd

G H

TS

Cp dH=dT T dS=dT

3 4 5

where T = temperature, DG0 = standard free energy change and R = gas constant. The free energy of protein–protein complex formation is linked to the equilibrium constant or affinity by eqn (3), so it is possible to estimate the DG0 value by determining the Kd value. The Kd values for protein–protein complexes are within the range 10 4–10 14 M which corresponds to DG values of 6–19 kcal/mol (Janin, 2000). The Gibbs free energy indicates the favourable direction of processes. Changes of Gibbs energy are related to changes of enthalpy (DH) and entropy (DS) [eqn (4)]. With respect to protein–protein interaction, this equation reflects two opposite tendencies, decrease of energy of the system and complex dissociation due to Brownian and intramolecular vibration motions. Change of enthalpy depends on hydrogen bond formation, electrostatic and van der Waals interactions, whereas the change of entropy component depends on changes of conformational freedom of the system. Conformational entropy is often subdivided into backbone and side chain contributions (Brady and Sharp, 1997). The backbone conformation entropy dominates in protein folding, but it has a modest contribution to protein– protein interactions when backbone changes are minor (Stites, 1997). The main contribution of the conformation entropy in the protein–protein interaction is usually the side chain component (Brady and Sharp, 1997). The solvent and association entropy are the other important components of J. Mol. Recognit. 2002; 15: 405–422


entropy. Protein–protein complex formation leads to release of water molecules from the surfaces of the protein interface into the solvent; this generally results in an increase of solvent entropy (Brady and Sharp, 1997). Protein complex formation is accompanied by a reduction of the translational and rotational freedom of partners that results in a change of association entropy (Brady and Sharp, 1997). When the net entropy change is positive, the protein–protein interaction is entropy-driven; in the opposite case, the enthalpy is the primary driving force of the interaction. Analysis of enthalpy and entropy changes of protein complex formation (69 complexes) demonstrates that, at physiological temperature, enthalpy favours protein–protein interaction in 74% of cases, while entropy favours formation in 55% of the complexes (Stites, 1997). At different temperatures, the leading driving force can be different. The interaction of hen egg white lysozyme and Fab D 1.3 was driven by enthalpy (at temperature below 23 °C), by enthalpy and entropy (between 23 and 35 °C), and only by entropy (above 35 °C; Zeden-Lutz et al., 1997). In most cases, the effects of enthalpy and entropy are opposite. This leads to enthalpy/ entropy compensation that results in small changes in DG values (Brady and Sharp, 1997). The description of protein–protein binding requires determination of the hydration states of hydrophobic groups at interfaces that is reflected in a change DCp (Janin, 1995). These groups become accessible to water molecules during complex dissociation. The translocation of the hydrophobic groups from water to non-polar environment is characterized by a large negative DCp value. Since in most protein– protein complexes the DCp values are negative, this indicates the vital importance of the hydrophobic interaction in protein complex formation (Stites, 1997). For several protein complexes a correlation between DCp and the values of hydrophobic interaction was found (Gomez and Freire, 1995). We showed that in electron-transport monooxygenase systems containing cytochrome P450 the hydrophobic force plays the major role in complex formation between membrane bound cytochrome P4502B4 and its redox partners cytochrome b5 and NADPH cytochrome P450 reductase. However it was shown that the electrostatic interaction reduces the rate of formation of these complexes, although it increases complex stability (Archakov and Ivanov, 1999). When proteins form tight complexes, kinetic measurement of Kd is preferable to equilibrium methods. The equilibrium constant (Kd) represents the ratio of dissociation (koff) and association (kon) rate constants. Typical association constants are in the range from 105 to 107 M 1 s 1 (Janin, 1995, 2000). The fastest complex formation rate was found for the interaction of barnase with barstar (2 109 M 1 s 1; Schreiber et al., 1997). The association rate can reach nearly diffusion-limited values (Gloss and Matthews, 1998; Janin, 1995). Typical Kd and koff values vary from 10 6 to 10 14 M, and from 103 to 10 7 s 1, respectively. For example, the lifetime of an antigen– antibody complex may be about a year (koff < 10 6 s 1; Yeung et al., 1995), whereas for protein complexes of the cytochrome P450 electron-transport system this parameter is limited to several minutes (Archakov and Ivanov, 1999; Ivanov et al., 1999b, 2001). Usually point mutations at interfaces reduce the affinity of the complex. As a rule a Copyright # 2002 John Wiley & Sons, Ltd.

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mutation results in increasing koff values and a minor effect on kon values (Janin, 2000; Schreiber et al., 1997). The relationship between equilibrium and rate constants depends on the mechanism of protein–protein interaction. The simplest mechanism is a one-step reaction, when proteins form a complex without conformational adaptation to each other [equation of reaction is the same as eqn (1)]. In a two-step mechanism, when complex formation is accompanied by conformational changes of monomers, the reaction may be written as: AB

0 k2 k1 ! AB ! AB k 2 1

6

k

where AB' is an intermediate complex before conformational changes. Then the Kd value is: Kd

k 1k 2 K1 K2 k1 k2

7

Usually k2 is larger than k 2 and this shifts the reaction to the right. When conformational changes are faster (in comparison to intermediate complex dissociation, i.e. k2 is large relative to k 1), eqn (6) reduces to eqn (1). A more complex situation appears when electron transfer proteins form protein–protein complexes. The minimal reaction scheme for bimolecular complex formation of oxidized electron acceptor and reduced electron donor with electron transfer reaction consists of five steps: association of proteins, equilibration of their energy levels, electron transfer, relaxation of protein complex of monomers with changed red-ox states and complex dissociation (Mathews et al., 2000). So the fit between kinetic and equilibrium data depends on a scheme that better represents the mechanism of protein–protein interaction. It is tempting to subdivide the process of protein–protein interaction into two possible mechanisms responsible for complex formation and stabilization. The first might underline the protein recognition followed by subsequent complex stabilization due to direct docking of protein monomers. In this case long-distance electrostatic forces determine the oriented factor. However, at this stage the thermodynamic barrier exists and the complex formation constant must be below the diffusion-limited constant (kD). The second mechanism represents only random collisions of the proteins monomers (kon → kD) with subsequent fixation of the complexes formed, which allows a high thermodynamic barrier to be overcome. Complex formation is especially favourable when kon → kD and koff → ?. In the case of permanent complexes the situation is much more complex because their formation appears to be a continuation of the folding of three-dimensional structures and cannot be evaluated by such simple thermodynamic considerations. Recognition requires the directed forces of interaction such as hydrogen bonds and electrostatic forces, whereas the binding energy is probably also determined by hydrophobic forces. Mutational analysis and analysis of the influence of ionic strength on the interaction of the T lymphocyte cell– cell recognition molecule CD2 with its ligand—CD48 showed little contribution of charged residues of the contacted surface of CD2 to binding energy of their interaction, whereas the loss of these charged residues leads to marked reduction of ligand-binding specificity (Davis et al., 1998). For the human growth hormone receptor J. Mol. Recognit. 2002; 15: 405–422

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complex, it was shown that the hydrophobic area plays the major role in the binding ability. Polar and charged residues surrounding this area are less important for binding (Wells, 1996). Eight amino acids (six of them are hydrophobic) out of 31 at the receptor interfaces of human growth hormone account for about 85% of the binding energy (Wells, 1996). The receptor site contains nine amino acids (six amino acids are hydrophobic) that can account for all the binding affinity (Clackson and Wells, 1995). Numerous directed-mutagenesis experiments demonstrate that in some cases mutation of only one amino acid destroys protein–protein binding (Aoki et al., 1998b; Behlke et al., 1998; Chen et al., 2000; Eubanks et al., 2000; Gomez-Moreno et al., 1998; Grant et al., 2000; Koltzscher and Gerke, 2000; Lin et al., 1999; Lomax et al., 1998; Martin et al., 1999a; Ramadevi et al., 1998; Saarela et al., 1998; Scott et al., 2000; Sengchanthalangsy et al., 1999; Sundberg et al., 2000; Thomas et al., 1998; Zeng et al., 1999), whereas in other cases only a few residues affect binding affinity (Clackson and Wells, 1995; Jackson, 1999; Pons et al., 1999; Wells, 1996) or complex stability (Martin et al., 1999b; Mateu and Fersht, 1998; Vaughan et al., 1999; Xie et al., 1999). It is suggested that most of the binding energy is related to only a few amino acids from the interface, so-called ‘hot spots’ (Bogan and Thorn, 1998; Bradshaw et al., 2000; Cunningham and Wells, 1997; Hu et al., 2000b; Kuhlmann et al., 2000; McInnes et al., 2000; von Kries et al., 2000). Thus, the same forces are involved in protein–protein interaction, protein folding and ligand–receptor interaction. The dominant factor for permanent complex formation is the hydrophobic force as for protein core folding. Thus such complex formation has some similarities with folding process. On the other hand, various forces participate in formation of the non-obligate complexes and those properties of the contact surfaces are more complex. Morphology of protein–protein interfaces So far we considered the properties and the driving forces of the protein–protein interaction without taking into consideration their spatial distributions, but examination of their average values cannot give the correct notion of the protein– protein interactions. The distribution of these properties is not chaotic and corresponds to their functional properties. The permanent complexes have hydrophobic surfaces at their interfaces, which differ insignificantly from the protein core (Tsai et al., 1997b). The morphology of the interfaces of the non-obligate complexes is more variable (Larsen et al., 1998) (Plate 1). Analysis of the morphology of protein– protein interfaces showed that one-third of protein complexes have interfaces with a well-defined hydrophobic core surrounded by a ring of polar groups [Larsen et al., 1998; Plate 1(A)]. The water molecules are usually located in these rings (Davies and Cohen, 1996; Larsen et al., 1998). Such interfaces were shown in several studies (Tochio et al., 1999; Wells, 1996). The other two-thirds of protein interfaces have mixed hydrophilic properties without a definite continuous hydrophobic patch [Larsen et al., 1998; plate 1(B)]. This type of protein interface has mixed short hydrophobic patches, polar groups and intersubunit hydrogen bonds (Larsen et al., 1998). Water molecules are located at the Copyright # 2002 John Wiley & Sons, Ltd.

protein interfaces, usually in cavities (Davies and Cohen, 1996; Larsen et al., 1998; Xu et al., 1997b). Monomers forming non-obligate complexes are either in polar solution or in the bound state in the complex. In the latter case they interact with each other, and their contact areas are shielded from the environment. In this case the hydrophobic surfaces are more optimal. At the same time, in the polar solution they should be hydrophilic enough to avoid non-specific aggregation, and whenever possible to shield the hydrophobic area of contact surface from the solvent. This is achieved by arrangement of the charged and polar groups around the hydrophobic area or by decreasing the large continuous hydrophobic patches by ‘dissemination’ of polar groups.

CHANGES OF PROTEIN–PROTEIN INTERACTIONS Since protein–protein interactions play a critical role in living systems, they are controlled by numerous intracellular mechanisms and physico-chemical factors. Temperature, ionic strength, pH and other physicochemical factors can influence protein–protein interactions. At high temperature, head shock protein 90 oligomerizes and shows a new chaperone activity (Yonehara et al., 1996). The ionic strength of solution can affect the oligomeric states of protein (Brazil et al., 1998; Shima et al., 1998). Ionic strength influences the kinetics of protein–protein interaction. For example, a decrease of ionic strength by one order of magnitude leads to the reduction to the same degree of kon and koff values of interaction of partners of cytochrome P450 monooxygenase systems (Archakov and Ivanov, 1999). This suggests the existence of a thermodynamic barrier both of the stage of complex formation and its dissociation. The stability of protein complexes also depends on pH (Gibas et al., 1997; Xie et al., 1998, 1999). The mechanisms responsible for these changes include charge masking (Gibas et al., 1997), conformational changes leading to hydrophobic surface exposure (Valentemesquita et al., 1998), and changes of association/dissociation rate equilibrium (Xie et al., 1998). Chelators (such as EDTA) can induce disintegration of a metal-containing complex in which metal ions are involved in complex formation (e.g. insulin; Murray-Rust et al., 1992). Some proteins form dimers via disulphide bridges. This process may occur spontaneously (Bell et al., 1998; Le et al., 2000; Pace et al., 1999) or during termoinactivation (Sasvari and Asboth, 1998) and free radical action (Kato et al., 2000). This process can be regulated by chaperones (Bass et al., 1998) and protein disulphide isomerases (Markus and Benezra, 1999). Cells can regulate the oligomerization state of their proteins by covalent modification. The latter includes phosphorylation (Behlke et al., 1998; Dare et al., 1999; Eisenmessers and Post, 1998), glycosylation (Bell et al., 1998; Tsuda et al., 2000), sulfation (Kehoe and Bertozzi, 2000), palmitoylation (Dunphy and Linder, 1998) and myristoylation (Taniguchi, 1999). A well-known example of regulation of the protein–protein interaction by covalent modification is phosphorylation in the signal transduction cascade (Eyster, 1998). J. Mol. Recognit. 2002; 15: 405–422


Plate 1. Examples of morphology of protein±protein interface. (A) Surface with continuous hydrophobic patch (Bence±Jones immunoglobulin; PDB1REI); (B) surface without continuous hydrophobic patch (endonuclease; PDB1SMN). Hydrophobicity increases in the following order: blue±green±brown.


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Figure 2. Structures of dimerizers, their anchors and linkers.

Another mechanism influencing protein–protein interaction is noncovalent ligand binding. The latter can induce formation (Laitinen et al., 2001; Ratovitski et al., 1999; Wells, 1996) or stabilization of protein complexes (Okada, 1998; Rafferty et al., 1999; Smyczynski et al., 2000), and also their dissociation (Rodriguezcrespo et al., 1998; Saito et al., 1998) or prevention of another complex formation (Garnier et al., 1998). Ligand binding can also lead to enzyme inhibition (Huang et al., 1997; Ju et al., 1998; Kojima et al., 1998). The most studied processes of the regulation of the protein–protein interaction by noncovalent ligand binding are receptor systems responsible for cell signalling (Klemm et al., 1998). For example, human growth hormone (hGH) induces dimerization of its receptor. At the first stage, hGH interacts with the monomeric form of its receptor. During the next stage, the second subunit of the receptor interacts with this complex and the resultant trimer complex becomes active and capable of transducing a signal (Wells, 1996). Such a mechanism explains the difference between the effect of agonists and antagonists. An agonist does induce a change in the conformation of receptor subunit leading to the receptor dimerization, whereas an antagonist does not (Abdalla et al., 1999; Wells, 1996). Thus, protein–protein interactions play an important role Copyright # 2002 John Wiley & Sons, Ltd.

in cells and they are regulated by different mechanisms. Therefore, artificial induction or disintegration of protein complexes can lead to different physiological responses of the cell. Over several years, different research groups have tried to design compounds which could directly influence protein–protein interactions. At the present moment, several compounds inducing protein dimerization (dimerizers; Austin et al., 1994; Clemons, 1999; Michnick, 2000), and compounds preventing dimer formation (inhibitors of dimerization, ID; Cochran, 2001; Way, 2000; Zutshi et al., 1998), have been discovered or designed.

ARTIFICIAL SYSTEMS Dimerizers Many cell-signalling pathways are initiated by protein– protein dimerization. So the main idea of dimerizer design consists in induction of such dimerization by small molecules and activation of a certain cell signal transduction pathway. Since dimerizers must interact with two separate proteins, they consist of three parts: two anchor groups interacting with the proteins and a long linker between them (Fig. 2). The chemical structure of each anchor group can be J. Mol. Recognit. 2002; 15: 405–422

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either identical (Amara et al., 1997; Blau et al., 1997; Keenan et al., 1998) or different (Belshaw et al., 1996). At present the anchor groups employed are FK506, rapamycin, cyclosporin, coumermycin and others (Austin et al., 1994; Belshaw et al., 1996; Clemons, 1999; Ho et al., 1996; Kopytek et al., 2000; Smith and Vanetten, 2001). Linkers consist of 5–16 atoms (Amara et al., 1997; Keenan et al., 1998). FK1012 is the most frequently used dimerizer. It is a non-toxic lipid-soluble dimer of the drug FK506. The latter selectively interacts with endogenous FK506-binding protein 12 (FKBP12) and this complex interacts with the cellular phosphatase, calcineurin, and inhibits it (Clemons, 1999). The concentrations required for induction of dimerization by such compounds vary within a relatively narrow range of 1–10 nM (Amara et al., 1997; MacCorkle et al., 1998). The effectiveness of dimerizers depends on the anchors affinity (Keenan et al., 1998) and the length of the linkers (Amara et al., 1997; Keenan et al., 1998). There were several attempts to design simple-analogues of FK506 (Clackson et al., 1998; Keenan et al., 1998). However, they were much less effective (Keenan et al., 1998). At present dimerizers are used only in laboratory practice to study cell proliferation (Blau et al., 1997), transcription (Amara et al., 1997; Belshaw et al., 1996), apoptosis (Amara et al., 1997; MacCorkle et al., 1998). However, these systems can be potentially used for clinical purposes, particularly in gene therapy. Dimerizers can be employed for induction of cell proliferation (Blau et al., 1997), or for elimination of transferred gene product and genetically modified cells by dimerizer-inducing apoptosis (Amara et al., 1997; MacCorkle et al., 1998). It is suggested to insert the additional chimeric gene, coding apoptosis-inducing protein with dimerizer-interacting domain, in genetically modified cells. When it is necessary to eliminate such modified cells, the dimerizers could be introduced into the cells. Since the dimerizer interacts with its target inducing apoptosis in the modified cells only, this will cause death of modified cells only (MacCorkle et al., 1998). Inhibitors of dimerization Numerous proteins act either as oligomer complexes, or they change aggregate states during their action. So inhibitors of dimerization (IDs) may prevent formation of an active dimer. Such inhibitors have been discovered for three HIV enzymes (protease, reversed transcriptase, invertase; Bouras et al., 1999; Morris et al., 1999; Sourgen et al., 1996; Zutshi et al., 1998), ribonucleotide reductase (Liuzzi et al., 1994) and DNA polymerase (Digard et al., 1995) of herpes simplex virus, human gluthatione reductase (Nordhoff et al., 1997), phosphatidylinisitol 3-kinase (Eaton et al., 1998), virus capsid (Hilpert et al., 1999; Prevelige, 1998) and some others (Beaulieu et al., 1999; Brickner and Chmielewski, 1998; Chen et al., 2001; Crump et al., 1998; Findeis, 2000; Gay et al., 1999b; Ghosh et al., 1999; Hart et al., 1999; Kim et al., 1999; Li et al., 1998; Lou et al., 1999; Pacofsky et al., 1998; Prasanna et al., 1998; Saito et al., 1998; Singh et al., 2001; Vu et al., 1999; Usui et al., 1998; Yao et al., 1998, 1999). Most of the discovered IDs are peptides resembling dimer interfaces (Zutshi et al., 1998), although peptidomimetic molecules or small organic molCopyright # 2002 John Wiley & Sons, Ltd.

ecules have also been found (Bouras et al., 1999; Findeis, 2000; Gay et al., 1999b; Sennequier et al., 1999; Souroujon and Mochly-Rosen, 1998; Yao et al., 1999; Zutshi et al., 1998). The activity of such inhibitors (expressed as IC50 or Ki values) varied from low nanomolar (Eaton et al., 1998; Schramm et al., 1999; Shultz and Chmielewski, 1999) to micromolar concentration range (Beaulieu et al., 1999; Sennequier et al., 1999; Usui et al., 1998; Yao et al., 1999; Zutshi et al., 1998). Peptide inhibitors of protein dimerization exhibit two important features. The longer peptides representing dimer interfaces are more potent inhibitors than their shorter derivatives (Digard et al., 1995; Ghosh and Chmielewski, 1998; Vu et al., 1999). Another important feature of peptide IDs is a possibility of improvement of their inhibitory activity by optimizing their amino acid composition and sequence. Using site-directed mutagenesis or methods of combinatorial chemistry more potent peptide inhibitors (than the primary peptide fragments from the dimer interface) have been developed (Hart et al., 1999; Li et al., 1998; Morris et al., 1999; Pacofsky et al., 1998; Shultz and Chmielewski, 1999; Zutshi et al., 1998). HIV protease is the most studied protein target for IDs (Ast et al., 1998; Bouras et al., 1999; Schramm et al., 1999; Shultz et al., 2000; Shultz and Chmielewski, 1997, 1999; Ulysse and Chmielewski, 1998; Zutshi et al., 1997, 1998). The active dimer of HIV protease is formed by interaction of two b-sheets from each subunit (Wlodawer, Vondrasek, 1998). It was initially found that peptides corresponding to the N- and C-termini of HIV protease inhibit its activity (Zutshi et al., 1997). Subsequently it was shown that some synthetic peptides are more potent inhibitors of HIV protease than those derived from the subunit interfaces (Ulysse and Chmielewski, 1998; Zutshi et al., 1997, 1998). The first generation of such inhibitors had flexible linkers (Shultz and Chmielewski, 1997; Ulysse and Chmielewski, 1998; Zutshi et al., 1997, 1998) with optimal distance ˚ (Zutshi et al., between peptides fragments of about 10 A 1998). The most potent inhibitor containing a flexible linker had an IC50 of 25 nM (Zutshi et al., 1997). Inhibitors with rigid linkers (‘molecular tongs’), containing tri- or tetrapeptidic arms attached to pyridinediol or naphthalenendiol were less active (Ki about 0.56–4.5 mM) (Bouras et al., 1999). High inhibitory potency (Ki in a low nanomolar range) was shown for lipopeptides containing peptide, linker and lipid (Schramm et al., 1999). Recently a nonpeptidic inhibitor of HIV-1 protease dimerization was designed (Song et al., 2001). In addition to ability to prevent protein dimerization, some IDs can also cause dimer dissociation. It was found that the imidazole derivative, clotrimazole, induced dissociation of inducible NO synthase into subunits in the absence of L-arginine and tertahydrobiopterin, whereas other derivatives only prevented dimerization (Sennequier et al., 1999). The fungal metabolite, tryprostatin A, induced reversible disruption of the cytoplasmic microtubule assembly in 3Y1cells (Usui et al., 1998). Thiol reagent 4,4'-dithiodipyridine, which covalently binds to cystein-458 of GroEL chaperonin, induced its dissociation (Bochkareva et al., 1999). The attacked cysteine is located in an almost inaccessible region between subunits of the protein and the ability of large a SH-reagent to interact with its target J. Mol. Recognit. 2002; 15: 405–422


suggests that during molecular dynamics this protein turns into an opened state. Peptides, which induce dissociation into monomers of HIV-1 protease and integrase, were found (Maroun et al., 2001; Park and Raines, 2000). Inhibition of protein(peptide)–receptor interactions Protein–protein interactions play the key role in receptormediated signal transduction. This process can be subdivided into several steps: interaction of the protein(peptide) hormones with their receptors followed by interaction of the hormone–receptor complex with other proteins of the signal transduction cascades. Modification of each of these interactions can be employed to change the cell metabolism. Design of inhibitors preventing receptor-mediated signal transduction may be similar to that of inhibitors of HIV protease dimer formation (see above). The design of interleukin-1 receptor antagonists was initiated by discovery of small (10–12 residues in length) peptides exhibiting modest inhibitory activity (IC50 about 10 5 M) (Yanofsky et al., 1996). The subsequent screening of a peptide library resulted in the discovery of 15 residue peptides with IC50 of about 2 nM. The substitution of proline for azetidine, aminoterminal acetylation and carboxy-terminal amidation gave peptidomimetic with IC50 about 0.5 nM (Cunningham and Wells, 1997). Other ways for the design of antagonists employed combinatorial chemistry, high-throughput screening and computer simulation methods. These methods were used for design of receptor antagonists for vascular endothelial growth factor (Aviezer et al., 2000), somatostatin (Rohrer et al., 1998), neuropeptide Y (Parker and Parker, 2000), thromboxane A2 (Marusawa et al., 1999), protease-activated receptors (Andrade-Gordon et al., 1999; Fujita et al., 1999; Hoekstra et al., 1998) and others (Beebe et al., 2000; Chackalamannil et al., 2001; Freidinger, 1999; Nicole et al., 2000; Yudt and Koide, 2001). There are many examples of successful design of effective nonpeptide ligands for different types of receptors of peptide(protein) hormones (Freidinger, 1999). On the basis of these results, peptidomimetics for these receptors were subdivided into three types (Ripka and Rich, 1998): peptidomimetics of the first type simulate the peptide backbone; peptidomimetics of the second type are small molecules that elicit agonist or antagonist activity, but they do not mimic the structure of native ligands; non-peptide molecules of the third type mimic the main binding elements of the peptide ligands. The ability to regulate signal transduction at the various steps by the inhibition of protein–protein interactions was shown using a signal cascade induced by light adsorption by rhodopsin. Retinal light absorption results in conformational change in the rhodopsin molecule and its interaction with the membrane bound G protein (Gt). The latter interacts with cGMP phosphodiesterase. Interaction of phosphorylated rhodopsin with arrestin (this protein blocks the interaction of rhodopsin with Gt) interrupts the signal transduction. Different peptides that can stop all stages of cascade induced by the light adsorption by rhodopsin were identified (Zutshi et al., 1998). Recently it was shown that many receptors coupled to Gprotein act as oligomer complexes (Overton and Blumer, Copyright # 2002 John Wiley & Sons, Ltd.

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2000; Salahpour et al., 2000) and peptides, the structural analogues of the receptor transmembrane domains, can destroy their interaction (Tarasova et al., 1999). Numerous investigations have been done in an attempt to design IDs for proteins possessing stable domains, which interact with receptors: SH2 domain (Alligood et al., 1998; Beaulieu et al., 1999; Burke et al., 1999, 2001; Cody et al., 2000; Davidson and Martin, 2000; Eaton et al., 1998; Fretz et al., 2000; Furet et al., 1999, 2000; Gao et al., 2000; Gay et al., 1999a,b; Garciaecheverria, 2001; Hart et al., 1999; Kim et al., 1999; Lou et al., 1999; Metcalf et al., 2000; Niimi et al., 2001; Pacofsky et al., 1998; Rickles et al., 1998; Schoepfer et al., 1998, 1999; Shakespeare, 2001; Shakespeare et al., 2000; Violette et al., 2000; Vu, 2000; Vu et al., 1999; Walker et al., 2000; Yao et al., 1999), SH3 domain (Dalgarno et al., 1997; Nguyen et al., 2000; Witter et al., 1998) and PDZ domain (Fuh et al., 2000).

FUTURE PERSPECTIVES Protein–protein interactions play a pivotal role in numerous cellular processes. The formation of permanent protein complexes can be considered as the extended folding of these proteins. Subunits of such proteins have similar structure, amino acid distribution over dimer interfaces and forces participating in the protein interaction as in the protein core (Srivastava and Sauer, 2000; Stites, 1997; Tamura and Privalov, 1997; Tsai et al., 1996). Final folding of subunits occurs during formation of such complexes (Srivastava and Sauer, 2000; Tsai et al., 1997b; Wallace and Dirr, 1999). However, the structure and properties of the protein interfaces of the non-obligate complexes have a dual nature. They share similarity with the protein core and resemble enzyme active site surfaces. High specificity of the interaction of these proteins suggest complementarity of dimer subunits and therefore distribution of their unique properties over protein interfaces. In this sense, they can be compared with active sites of enzymes, which are also influenced by their substrates. For example, protein–protein interaction and binding of peptide inhibitors to the active site of protease represent the same process (Wlodawer and Vondrasek, 1998). So the protein interfaces of the subunits of the non-obligate complexes can be considered to some extent as the equivalent of enzyme active sites. They represent protein regions for which new biologically active compounds, in particular drugs, can be designed. Numerous results during recent years unquestionably support validity of such an approach. Successful examples have been found using different cells and enzymes. The designed compounds can act as dimerizers (Austin et al., 1994; Clemons, 1999), and also as agents preventing this process (inhibitors of dimerization and antagonists of peptide/protein receptors; Beeley, 2000; Cochran, 2000, 2001; Cody et al., 2000; Freidinger, 1999; Way, 2000; Zutshi et al., 1998). Many designed compounds possess high activity and selectivity. Dimerizers are also used to change cell functions. This may be achieved by acting at various systems. Therefore at present their employment in clinical practice is limited by our insufficient knowledge about these processes. Since dimerizers consist of three parts, they are rather large J. Mol. Recognit. 2002; 15: 405–422

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molecules, and their transport to a particular destination through numerous cellular and tissue barriers may represent a serious problem. At the same time, the universality of such systems may have important advantages. The same dimerizer can be used for many purposes since its action depends on the constructed target (original protein with fused dimerizer binding domain). The further development of this field will probably be focused on detailed characterization of the cell systems, which can be modified by dimerizers, and in designing new dimerizers (the binding parts and linkers) for increasing selectivity for existing domain(s) or discovering new ones, in particular, for design, the anchor part for mutant binding domain to exclude a capability of dimerizer binding to normal cell proteins containing such a domain (Clackson et al., 1998; Yang et al., 2000). Protein–protein interactions have big potential as a new class of targets for the novel generation of drugs. However, the search for new potential targets for these drugs requires some common criteria. In our viewpoint, these criteria must include the following points: (1) Low-molecular-weight compounds are more effective when the sizes of contacted interfaces are quite small. In this case we may expect that the binding energy of proteins is not high, and the small molecule can interact with a considerable part of these contact surfaces. (2) IDs should preferably interact with amino acids representing ‘hot spots’ of the interface. In this case the interaction of IDs with such amino acids would effectively prevent protein complex formation. The inhibitors of dimerization can be applied both for modification of regulator processes (as dimerizers), and for prevention of formation of the enzyme active form. Methods for IDs design can be the same as for inhibitors binding at the active site of an enzyme. These include experimental methods [combinatorial chemistry (Beebe et al., 2000; Hart et al., 1999; Nefzy et al., 1998), highthroughput screening (Lebl, 1999)], computer approaches [molecular database mining (Loughney et al., 1999; Marrone et al., 1997), computer-aided design (Fretz et al., 2000; Marrone et al., 1997; Schoepfer et al., 1998; Zeng, 2000)] and others. The classic pathway for drug design (peptide–peptidomimetic–small organic molecule) is also applicable (Beeley, 2000; Liu, 1999; Stigers et al., 1999; Vu, 2000) and several positive results have recently been reported (Alligood et al., 1998; Beaulieu et al., 1999; Cunningham and Wells, 1997; Freidinger, 1999; Hart et al., 1999; Schoepfer et al., 1998; Witter et al., 1998). Recently, a new approach for the first step of ID design has been proposed. It requires the development of a single chain antibody against one interaction surface of one protein and use of this antibody as a template for design of inhibitors (Chrunyk et al., 2000). Another important question in IDs design is the possibility of obtaining relatively small compounds, since peptides are not optimal for medicinal practice. ‘Unlike the interactions of enzymes with non-protein substrates, protein–protein interactions usually do not occur in tightly binding pockets. Instead, interactions between proteins Copyright # 2002 John Wiley & Sons, Ltd.

usually occur across large, flat surfaces’ (Way, 2000); however, ‘most studies show that relatively few residues within these large contact surfaces actually drive binding’ (Cunningham and Wells, 1997). Furthermore, it was noted that in several studies a mutation of one amino acid leads to full loss of protein dimerization ability or to significant decrease of binding constants (Behlke et al., 1998; Eubanks et al., 2000; Imai et al., 2000; Koltzscher and Gerke, 2000; Lin et al., 1999; Lomax et al., 1998; Martin et al., 1999a; Omata et al., 2000; Ramadevi et al., 1998; Saarela et al., 1998; Sengchanthalangsy et al., 1999; Stenberg et al., 2000; Thomas et al., 1998; Zeng et al., 1999). Also oxidation of two methionine residues to methionine sulphoxide at the dimer interface of HIV-2 protease resulted in inactivation of this enzyme (Davis et al., 2000). There are some indications that the IDs, interacting with such critical amino acids, may be relatively small molecules. Nevertheless, the problem for small IDs still exists. Recently, a new strategy to design IDs to increase the effectivity of such inhibitors has been proposed. At the first step, ID interacts with its target non-covalently. This interaction brings together weakly reactive groups of the drug and amino acid side chain. At the second step, such contacted groups covalently interact with each other (Way, 2000). The same strategy was used to design inhibitors of dimerization of HIV protease and SH2 domain (Violette et al., 2000; Zutshi and Chmielewski, 2000). The designed molecules form disulphide bridges between drugs and cysteine located at protein interface surfaces. It is possible to assume that IDs can be a useful class of compounds for design of antibiotic, antiviral and parasitic drugs. There are two favourable features of IDs for such drugs. Design of potential antibiotic interacting with the active site of enzyme can be limited by the high structural conservation of active sites, which can prevent an effective distinction between the human and the pathogen enzymes. The greater structural variability of protein–protein interfaces may supply a target for the effective differentiation between the host and pathogen enzymes (Singh et al., 2001). The second favourable feature of IDs is related to the problem of overcoming antibiotic resistance of pathogens. One of the preferential mechanisms of antibiotic resistance is the mutation of amino acid in the active site of the antibiotic-target enzyme. Since the substrate and drugs can interact with the different groups at the active site, the mutation of active site amino acid (but not catalytic residues) can lead to decreased affinity of the drug with little effect on substrate binding and enzyme activity. Numerous data exist that a single mutation in one subunit of protein–protein interface can destroy the protein–protein interaction (see above). The conservation of protein complex in this case requires the complementary mutation in the second subunit. The simultaneous double mutation in different subunits is much more unlikely, so it seems that the essential amino acids of protein–protein interface are quite conservative. It is also unlikely that pathogens acquire resistance for IDs binding to such amino acid residues. Recently, several compounds were found that may act as inhibitors of dimerization and as dimerizers. It was shown that some pyrrolidine derivatives are competitive inhibitors of serum amyloid P component (SAP) glycoprotein binding to amyloid fibrils. These low-weight compounds are also J. Mol. Recognit. 2002; 15: 405–422


able to dimerize SAP molecules, leading to their rapid elimination by the liver (Pepys et al., 2002). Finally, one more speculation can be made. Since small molecules can prevent the protein–protein interaction, it might be possible to design (or discover) small compounds that selectively inhibit protein folding. At present, no experimental data exist, but it may become an interesting field in drug design in the recent future. All of this gives hopes that in the future the compounds regulating protein–protein interactions will take a respect-

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able place adjacent to ‘classic’ drugs that interact with active sites of enzymes.

Acknowledgements This work was partially supported by the Russian Foundation for Basic Research (grants 01-04-48128, 99-04-48081) and a cooperation with ‘Janssen Pharmaceutica NY. Center for molecular design’. The authors thank Dr A. E. Medvedev for valuable and helpful discussions.

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