VEGFR2活性腔性质以及与抑制剂的结合模式研究

VEGFR2介导肿瘤诱导的血管生成作用, 是抑制肿瘤生长和转移的新靶点. 为深入探讨VEGFR2活性腔性质以及与抑制剂的结合模式, 采用多拷贝同时搜寻法(MCSS)研究VEGFR2活性腔的性质, 然后用分子对接方法对5个已上临床的VEGFR抑制剂与VEGFR2活性腔进行对接计算, 讨论它们的结合模式, 确定与配体结合相关的关键残基. 研究发现: 疏水腔I, II是配体结合的关键区域, 残基Glu915, Cys917是关键的氢键作用位点, Lys866, Glu883和Asp1044形成的极性区域对提高配体亲合力很重要, 疏水腔III和极性腔IV是额外增强配体结合力的区域, IV区的Arg1030可提供额外的氢键作用位点. 本研究可为全新VEGFR2抑制剂的合理药物设计提供理论依据, 为寻找新的抗肿瘤药物奠定基础.     

Homology modeling and molecular dynamics simulation of N-myristoyltransferase from protozoan parasites: active site characterization and insights into rational inhibitor design

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is a cytosolic monomeric enzyme that catalyzes the transfer of the myristoyl group from myristoyl-CoA to the N-terminal glycine of a number of eukaryotic cellular and viral proteins. Recent experimental data suggest NMT from parasites could be a promising new target for the design of novel antiparasitic agents with new mode of action. However, the active site topology and inhibitor specificity of these enzymes remain unclear. In this study, three-dimensional models of NMT from Plasmodium falciparum (PfNMT), Leishmania major (LmNMT) and Trypanosoma brucei (TbNMT) were constructed on the basis of the crystal structures of fungal NMTs using homology modeling method. The models were further refined by energy minimization and molecular dynamics simulations. The active sites of PfNMT, LmNMT and TbNMT were characterized by multiple copy simultaneous search (MCSS). MCSS functional maps reveal that PfNMT, LmNMT and TbNMT share a similar active site topology, which is defined by two hydrophobic pockets, a hydrogen-bonding (HB) pocket, a negatively-charged HB pocket and a positively-charged HB pocket. Flexible docking approaches were then employed to dock known inhibitors into the active site of PfNMT. The binding mode, structure–activity relationships and selectivity of inhibitors were investigated in detail. From the results of molecular modeling, the active site architecture and certain key residues responsible for inhibitor binding were identified, which provided insights for the design of novel inhibitors of parasitic NMTs.     

Structural analysis of charge discrimination in the binding of inhibitors to human carbonic anhydrases I and II

Despite the similarity in the active site pockets of carbonic anhydrase (CA) isozymes I and II, the binding affinities of benzenesulfonamide inhibitors are invariably higher with CA II as compared to CA I. To explore the structural basis of this molecular recognition phenomenon, we have designed and synthesized simple benzenesulfonamide inhibitors substituted at the para position with positively charged, negatively charged, and neutral functional groups, and we have determined the affinities and X-ray crystal structures of their enzyme complexes. The para-substituents are designed to bind in the midsection of the 15 A deep active site cleft, where interactions with enzyme residues and solvent molecules are possible. We find that a para-substituted positively charged amino group is more poorly tolerated in the active site of CA I compared with CA II. In contrast, a para-substituted negatively charged carboxylate substituent is tolerated equally well in the active sites of both CA isozymes. Notably, enzyme-inhibitor affinity increases upon neutralization of inhibitor charged groups by amidation or esterification. These results inform the design of short molecular linkers connecting the benzenesulfonamide group and a para-substituted tail group in "two-prong" CA inhibitors: an optimal linker segment will be electronically neutral, yet capable of engaging in at least some hydrogen bond interactions with protein residues and/or solvent. Microcalorimetric data reveal that inhibitor binding to CA I is enthalpically less favorable and entropically more favorable than inhibitor binding to CA II. This contrasting behavior may arise in part from differences in active site desolvation and the conformational entropy of inhibitor binding to each isozyme active site.     

Molecular recognition at the active site of factor Xa: cation-π interactions, stacking on planar peptide surfaces, and replacement of structural water

Factor Xa, a serine protease from the blood coagulation cascade, is an ideal enzyme for molecular recognition studies, as its active site is highly shape-persistent and features distinct, concave sub-pockets. We developed a family of non-peptidic, small-molecule inhibitors with a central tricyclic core orienting a neutral heterocyclic substituent into the S1 pocket and a quaternary ammonium ion into the aromatic box in the S4 pocket. The substituents were systematically varied to investigate cation-π interactions in the S4 pocket, optimal heterocyclic stacking on the flat peptide walls lining the S1 pocket, and potential water replacements in both the S1 and the S4 pockets. Structure-activity relationships were established to reveal and quantify contributions to the binding free enthalpy, resulting from single-atom replacements or positional changes in the ligands. A series of high-affinity ligands with inhibitory constants down to K(i)=2 nM were obtained and their proposed binding geometries confirmed by X-ray co-crystal structures of protein-ligand complexes.     

Two-prong inhibitors for human carbonic anhydrase II

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumvent such constraints, we contemplated enhancing the binding affinities of inhibitors by attaching tether groups, which would interact with the surface exposed amino acid residues. This strategy has been tested for the inhibition of human carbonic anhydrase II. Benzenesulfonamide serves as a weak inhibitor for the enzyme, but when it is conjugated to iminodiacetate-Cu2+ (which interacts with the surface-exposed His residues) via a spacer group, its binding affinity is enhanced by about 2 orders of magnitude. This "two-prong" approach is expected to serve as a general strategy for converting weak inhibitors of enzymes into tight-binding inhibitors.     

Design and pharmacophoric identification of flavonoid scaffold-based aromatase inhibitors

Aromatase is a crucial enzyme for the catalysis of aromatization reaction at the last and rate-limiting step involved in the conversion of androgenic substrates to an estrogenic substrate. A hormone-dependent breast cancer in postmenopausal woman can be cured by inhibition of estrogen biosynthesis by the help of aromatase inhibitors (AIs). The mode of interactions of flavonones with the active site of aromatase has been studied in search of potent and selective AIs as a substitute of the natural steroidal ligand. Structure-based computational approach namely, molecular docking simulations were performed to investigate the structural features of the docked complex of aromatase and flavonoid ligands. A nonsteroidal flavonoid pharmacophore showing electrostatic and steric features for selective binding within the main pocket of the catalytic active site of aromatase has been identified as an outcome of the study. The binding affinity of quercetin and isoflavone were predicted within aromatase. Isoflavone was used as a negative control to compare its binding affinities with the selected dataset. The predicted binding affinity of negative control isoflavone was in accordance with its in vitro AI efficacy. Isoflavone showed poor binding affinity and ranked last in terms of MolDock score (−86.309 kcal/molÅ) compared to dataset molecules. The generated pharmacophoric information will be helpful for the synthetic chemist to design and synthesize selective AIs with comparable binding affinity to the natural steroidal ligand.     

Crystallographic structures of two bisphosphonate:1-deoxyxylulose-5-phosphate reductoisomerase complexes

We have obtained the single-crystal X-ray crystallographic structures of the bisphosphonates [(1-isoquinolinylamino)methylene]-1,1-bisphosphonate and [[(5-chloro-2-pyridinyl)amino]methylene]-1,1-bisphosphonate, bound to the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase (DXR, EC 1.1.1.267, also known as 2-C-methyl-d-erythritol-4-phosphate synthase), an important target for the development of antimalarial drugs. Our results indicate that both bisphosphonates bind into the fosmidomycin binding site. The aromatic groups are in a shallow hydrophobic pocket, and the phosphonate groups are involved in electrostatic interactions with Mg2+ or a cluster of carboxylic acid groups and lysine while the fosmidomycin phosphonate-binding site is occupied by a sulfate ion (as also observed in the DXR/NADP+ structure). The availability of these two new crystal structures opens up the possibility of the further development of bisphosphonates and related systems as DXR inhibitors and, potentially, as antiinfective agents.     

Structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin

BACKGROUND: The aspartic proteinase renin plays an important physiological role in the regulation of blood pressure. It catalyses the first step in the conversion of angiotensinogen to the hormone angiotensin II. In the past, potent peptide inhibitors of renin have been developed, but none of these compounds has made it to the end of clinical trials. Our primary aim was to develop novel nonpeptide inhibitors. Based on the available structural information concerning renin-substrate interactions, we synthesized inhibitors in which the peptide portion was replaced by lipophilic moieties that interact with the large hydrophobic S1/S3-binding pocket in renin. RESULTS: Crystal structure analysis of renin-inhibitor complexes combined with computational methods were employed in the medicinal-chemistry optimisation process. Structure analysis revealed that the newly designed inhibitors bind as predicted to the S1/S3 pocket. In addition, however, these compounds interact with a hitherto unrecognised large, distinct, sub-pocket of the enzyme that extends from the S3-binding site towards the hydrophobic core of the enzyme. Binding to this S3(sp) sub-pocket was essential for high binding affinity. This unprecedented binding mode guided the drug-design process in which the mostly hydrophobic interactions within subsite S3(sp) were optimised. CONCLUSIONS: Our design approach led to compounds with high in vitro affinity and specificity for renin, favourable bioavailability and excellent oral efficacy in lowering blood pressure in primates. These renin inhibitors are therefore potential therapeutic agents for the treatment of hypertension and related cardiovascular diseases.     

Uracil-directed ligand tethering: an efficient strategy for uracil DNA glycosylase (UNG) inhibitor development

Uracil DNA glycosylase (UNG) is an important DNA repair enzyme that recognizes and excises uracil bases in DNA using an extrahelical recognition mechanism. It is emerging as a desirable target for small-molecule inhibitors given its key role in a wide range of biological processes including the generation of antibody diversity, DNA replication in a number of viruses, and the formation of DNA strand breaks during anticancer drug therapy. To accelerate the discovery of inhibitors of UNG we have developed a uracil-directed ligand tethering strategy. In this efficient approach, a uracil aldehyde ligand is tethered via alkyloxyamine linker chemistry to a diverse array of aldehyde binding elements. Thus, the mechanism of extrahelical recognition of the uracil ligand is exploited to target the UNG active site, and alkyloxyamine linker tethering is used to randomly explore peripheral binding pockets. Since no compound purification is required, this approach rapidly identified the first small-molecule inhibitors of human UNG with micromolar to submicromolar binding affinities. In a surprising result, these uracil-based ligands are found not only to bind to the active site but also to bind to a second uncompetitive site. The weaker uncompetitive site suggests the existence of a transient binding site for uracil during the multistep extrahelical recognition mechanism. This very general inhibitor design strategy can be easily adapted to target other enzymes that recognize nucleobases, including other DNA repair enzymes that recognize other types of extrahelical DNA bases.     

A proposed model of Mycobacterium avium complex dihydrofolate reductase and its utility for drug design

A homology model of Mycobacterium avium complex dihydrofolate reductase (MAC DHFR) was constructed on the basis of the X-ray crystal structure of Mycobacterium tuberculosis (Mtb) DHFR. The homology searching of the MAC DHFR resulted in the identification of the Mtb DHFR structure (PDB 1DF7) as the template for the model building. The MAC enzyme sequence was aligned to that of the Mtb counterpart using a modified Needleman and Wunsch methodology. The initial geometry to be modeled was copied from the template, either fully or partially depending on whether the residues were conserved or not, respectively. Using a randomized modeling procedure, 10 independent models of the target protein were built. The cartesian average of all the model structures was then refined using molecular mechanics. The resulting model was assessed for stereochemical quality using a Ramachandran plot and by analyzing the consistency of the model with the experimental data. The structurally and functionally important residues were identified from the model. Further, 5-deazapteridines recently reported as inhibitors of MAC DHFR were docked into the active site of the developed model. All the seven inhibitors used in the docking study have a similar docking mode at the active site. The network of hydrogen bonds around the 2,4-diamino-5-deazapteridine ring was found to be crucial for the binding of the inhibitors with the active site residues. The 5-methyl group of the inhibitors was located in a narrow hydrophobic pocket at the bottom of the active site. The relative values of the three torsion angles of the inhibitors were found to be important for the proper orientation of the inhibitor functional groups into the active site.     

Inhibition of protein interactions: co-crystalized protein–protein interfaces are nearly as good as holo proteins in rigid-body ligand docking

Modulating protein interaction pathways may lead to the cure of many diseases. Known protein–protein inhibitors bind to large pockets on the protein–protein interface. Such large pockets are detected also in the protein–protein complexes without known inhibitors, making such complexes potentially druggable. The inhibitor-binding site is primary defined by the side chains that form the largest pocket in the protein-bound conformation. Low-resolution ligand docking shows that the success rate for the protein-bound conformation is close to the one for the ligand-bound conformation, and significantly higher than for the apo conformation. The conformational change on the protein interface upon binding to the other protein results in a pocket employed by the ligand when it binds to that interface. This proof-of-concept study suggests that rather than using computational pocket-opening procedures, one can opt for an experimentally determined structure of the target co-crystallized protein–protein complex as a starting point for drug design.     

Exploring the active site of amine:pyruvate aminotransferase on the basis of the substrate structure-reactivity relationship: how the enzyme controls substrate specificity and stereoselectivity

An active site model of the amine:pyruvate aminotransferase (APA) from Vibrio fluvialis JS17 was constructed on the basis of the relationship between substrate structure and reactivity. Due to the broad substrate specificity of the APA, various amino donors (chiral and achiral amine, amino acid, and amino acid derivative) and amino acceptors (keto acid, keto ester, aldehyde, and ketone) were used to explore the active site structure. The result suggested a two-binding site model consisting of two pockets, one large (L) and the other small (S). The difference in the size of each binding pocket and strong repulsion for a carboxylate in the S pocket were key determinants to control its substrate specificity and stereoselectivity. The L pocket showed dual recognition mode for both hydrophobic and carboxyl groups as observed in the side-chain pockets of aspartate aminotransferase and aromatic aminotransferase. Comparison of the model with those of other aminotransferases revealed that the L and S pockets corresponded to carboxylate trap and side-chain pocket, respectively. The active site model successfully explains the observed substrate specificity as well as the stereoselectivity of the APA.     

Computational fragment-based drug design to explore the hydrophobic sub-pocket of the mitotic kinesin Eg5 allosteric binding site

Eg5, a mitotic kinesin exclusively involved in the formation and function of the mitotic spindle has attracted interest as an anticancer drug target. Eg5 is co-crystallized with several inhibitors bound to its allosteric binding pocket. Each of these occupies a pocket formed by loop 5/helix α2 (L5/α2). Recently designed inhibitors additionally occupy a hydrophobic pocket of this site. The goal of the present study was to explore this hydrophobic pocket with our MED-SuMo fragment-based protocol, and thus discover novel chemical structures that might bind as inhibitors. The MED-SuMo software is able to compare and superimpose similar interaction surfaces upon the whole protein data bank (PDB). In a fragment-based protocol, MED-SuMo retrieves MED-Portions that encode protein-fragment binding sites and are derived from cross-mining protein-ligand structures with libraries of small molecules. Furthermore we have excluded intra-family MED-Portions derived from Eg5 ligands that occupy the hydrophobic pocket and predicted new potential ligands by hybridization that would fill simultaneously both pockets. Some of the latter having original scaffolds and substituents in the hydrophobic pocket are identified in libraries of synthetically accessible molecules by the MED-Search software. Ksenia Oguievetskaia and Laetitia Martin-Chanas contributed equally to this work.     

Calculation of relative binding affinities of fructose 1,6-bisphosphatase mutants with adenosine monophosphate using free energy perturbation method

The free energy perturbation (FEP) methodology is the most accurate means of estimating relative binding affinities between inhibitors and protein variants. In this article, the importance of hydrophobic and hydrophilic residues to the binding of adenosine monophosphate (AMP) to the fructose 1,6-bisphosphatase (FBPase), a target enzyme for type-II diabetes, was examined by FEP method. Five mutations were made to the FBPase enzyme with AMP inhibitor bound: 113Tyr --> 113Phe, 31Thr --> 31Ala, 31Thr --> 31Ser, 177Met --> 177Ala, and 30Leu --> 30Phe. These mutations test the strength of hydrogen bonds and van der Waals interactions between the ligand and enzyme. The calculated relative free energies indicated that: 113Tyr and 31Thr play an important role, each via two hydrogen bonds affecting the binding affinity of inhibitor AMP to FBPase, and any changes in these hydrogen bonds due to mutations on the protein will have significant effect on the binding affinity of AMP to FBPase, consistent to experimental results. Also, the free energy calculations clearly show that the hydrophilic interactions are more important than the hydrophobic interactions of the binding pocket of FBPase.     

白色念珠菌N-肉豆蔻酰基转移酶中药物结合位点的MCSS分析

采用多拷贝同时搜寻方法(MCSS)分析得到了CaNMT活性位点的疏水区域、氢键结合位点和负电性区域. MCSS计算结果显示, CaNMT活性位点有两个疏水性比较强的区域: 一个由Tyr107, Tyr109, Val108, Phe117, Phe123, Ala127, Phe176和Leu337等残基组成; 另一个由Phe115, Phe240和Phe339组成. CaNMT活性位点发现有两个氢键作用区域, 其中Tyr119, His227, Asn392和Leu451是与已有抑制剂的氢键结合位点, Tyr107, Asn175, Thr211和Asp412是新发现的氢键结合位点, 而且在NMT家族中高度稳定, 它们对设计新结构类型的CaNMT抑制剂具有重要作用. Leu451是负电性兼氢键作用位点, 是抑制剂设计时所必需考虑的位点.     

Heterocyclic Substitutions Greatly Improve Affinity and Stability of Folic Acid towards FRα. an In Silico Insight

Folate receptor alpha (FRα) is known as a biological marker for many cancers due to its overexpression in cancerous epithelial tissue. The folic acid (FA) binding affinity to the FRα active site provides a basis for designing more specific targets for FRα. Heterocyclic rings have been shown to interact with many receptors and are important to the metabolism and biological processes within the body. Nineteen FA analogs with substitution with various heterocyclic rings were designed to have higher affinity toward FRα. Molecular docking was used to study the binding affinity of designed analogs compared to FA, methotrexate (MTX), and pemetrexed (PTX). Out of 19 FA analogs, analogs with a tetrazole ring (FOL03) and benzothiophene ring (FOL08) showed the most negative binding energy and were able to interact with ASP81 and SER174 through hydrogen bonds and hydrophobic interactions with amino acids of the active site. Hence, 100 ns molecular dynamics (MD) simulations were carried out for FOL03, FOL08 compared to FA, MTX, and PTX. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of FOL03 and FOL08 showed an apparent convergence similar to that of FA, and both of them entered the binding pocket (active site) from the pteridine part, while the glutamic part was stuck at the FRα pocket entrance during the MD simulations. Molecular mechanics Poisson-Boltzmann surface accessible (MM-PBSA) and H-bond analysis revealed that FOL03 and FOL08 created more negative free binding and electrostatic energy compared to FA and PTX, and both formed stronger H-bond interactions with ASP81 than FA with excellent H-bond profiles that led them to become bound tightly in the pocket. In addition, pocket volume calculations showed that the volumes of active site for FOL03 and FOL08 inside the FRα pocket were smaller than the FA–FRα system, indicating strong interactions between the protein active site residues with these new FA analogs compared to FA during the MD simulations.     

Comparative protein modeling of 1-deoxy-D-xylulose-5-phosphate reductoisomerase enzyme from Plasmodium falciparum: a potential target for antimalarial drug discovery

Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Pf-DXR) is a potential target for antimalarial chemotherapy. The three-dimensional model (3D) of this enzyme was determined by means of comparative modeling through multiple alignment followed by intensive optimization, minimization, and validation. The resulting model demonstrates a reasonable topology as gauged from the Ramachandran plot and acceptable three-dimensional structure compatibility as assessed by the Profiles-3D score. The modeled monomeric subunit consists of three domains: (1) N-terminal NADPH binding domain, (2) connective or linker domain (with most of the active site residues located in this domain), and (3) a C-terminal domain. This structure proved to be consistent with known DXR crystal structures from other species. The predicted active site compared favorably with those of the templates and appears to have an active site with a highly conserved architecture. Additionally, the model explains several site-directed mutagenesis data. Besides using several protein structure-checking programs to validate the model, a set of known inhibitors of DXR were also docked into the active site of the modeled Pf-DXR. The docked scores correlated reasonably well with experimental pIC50 values with a regression coefficient (R2) equal to 0.84. Results of the current study should prove useful in the early design and development of inhibitors by either de novo drug design or virtual screening of large small-molecule databases leading to development of new antimalarial agents.     

Synthesis of Novel Nonpeptidic Thrombin Inhibitors

A novel class of nonpeptidic, active, and selective thrombin inhibitors has resulted from X‐ray‐structure‐based design and subsequent improvement of the initial lead molecules. These inhibitors possess a bi‐ or tricyclic central core structure with attached side chains to reach the three binding pockets (selectivity S1 pocket, distal D pocket, and proximal P pocket) present in the active site of the enzyme. The key step in the preparation of these compounds is the 1,3‐dipolar cycloaddition between an azomethine ylide, prepared in situ by the decarboxylative method from an aromatic aldehyde and an α‐amino acid, with an N‐substituted maleimide (e.g., see Schemes 1 and 2). All potent inhibitors contain an amidinium residue in the side chain for incorporation into the S1 pocket, which was introduced in the last step of the synthesis by a Pinner reaction. The compounds were tested in biological assays for activity against thrombin and the related serine protease trypsin. The first‐generation lead compounds (±)‐ 11 and (±)‐ 19 (Scheme 1) with a bicyclic central scaffold showed K_i values for thrombin inhibition of 18 μM and 0.67 μM , respectively. Conformationally more restricted second‐generation analogs (Scheme 2) were more active ((±)‐ 22i : K_i=90 nM (Table 1)); yet the selectivity for thrombin over trypsin remained weak. In the third‐generation compounds, a small lipophilic side chain for incorporation into the hydrophobic P pocket was introduced (Schemes 7 and 8). Since this pocket is present in thrombin but not in trypsin, an increase in binding affinity was accompanied by an increase in selectivity for thrombin over trypsin. The most selective inhibitor (K_i=13 nM , 760‐fold selectivity for thrombin over trypsin; Table 2) was (±)‐ 1 with an i‐Pr group for incorporation into the P pocket. Optical resolution of (±)‐ 1 (Scheme 9) provided (+)‐ 1 with a K_i value of 7 nM and a 740‐fold selectivity, whereas (−)‐ 1 was 800‐fold less active (K_i=5.6 μM , 21‐fold selectivity). The absolute configuration of the stronger‐binding enantiomer was assigned based on the X‐ray crystal structure of the complex formed between thrombin and this inhibitor. Compound (+)‐ 1 mimics the natural thrombin substrate, fibrinogen, which binds to the enzyme with the Ph group of a phenylalanine (piperonyl in (+)‐ 1 ) in the distal D pocket, with the i‐Pr group of a valine (i‐Pr in (+)‐ 1 ) in the proximal P pocket, and with a guanidinium side chain of an arginine residue (phenylamidinium group in (+)‐ 1 ) in the selectivity S1 pocket of thrombin. A series of analogs of (±)‐ 1 with the phenylamidinium group replaced by aromatic and aliphatic rings bearing OH or NH₂ groups (Schemes 10 – 14) were not effectively bound by thrombin. A number of X‐ray crystal‐structure analyses of free inhibitors confirmed the high degree of preorganization of these compounds in the unbound state. Since all inhibitors prefer similar modes of association with thrombin, detailed information on the strength of individual intermolecular bonding interactions and their incremental contribution to the overall free energy of complexation was generated in correlative binding and X‐ray studies. The present study demonstrates that defined mutations in highly preorganized inhibitors provide an attractive alternative to site‐directed mutagenesis in exploring molecular‐recognition phenomena at enzyme active sites.     

Free energy perturbation calculations on models of active sites: Applications to adenosine deaminase inhibitors

Free energy perturbation calculations were performed to determine the free energy of binding associated with the presence of perhaps an unusual hydroxyl group in the transition state analog of nebularine, an inhibitor of the enzyme adenosine deaminase. The presence of a single hydroxyl group in this inhibitor has been found to contribute ?9.8 kcal/mol to the free energy of binding, with a 10⁸-fold increase in the binding affinity by the enzyme. In this work, we calculate the difference in solvation free energy for the 1,6-dihydropurine complex versus that of the 6-hydroxyl-1,6-dihydropurine complex to determine if this marked increase in binding affinity is attributed to an unusually hydrophobic hydroxyl group. The calculated ΔG associated for the solvation free energy is ?11.8 kcal/mol. This large change in the solvation free energy suggests that this hydroxyl is instead unusually hydrophilic and that the difference in free energy of interaction for the two inhibitors to the enzyme must be at least ca. 20 kcal/mol. Although the crystal structure for adenosine deaminase is currently not known, we attempt to mimic the nature of the active site by constructing models which simulate the enzyme-inhibitor complex. We present a first attempt at determining the change in free energy of binding for a system in which structural data for the enzyme is incomplete. To do this, we construct what we believe is a minimal model of the binding between adenosine deaminase and an inhibitor. The active site is simulated as a single charged carboxyl group which can form a hydrogen bond with the hydroxyl group of the analog. Two different carboxyl anion models are used. In the first model, the association is modeled between an acetic acid anion and the modified inhibitor. The second model consists of a hydrophobic amino acid pocket with an interior Glu residue in the active site. From these models we calculate the change in free energy of association and the overall change in free energy of binding. We calculate the free energies of interaction both in the absence and presence of water. We conclude from this that the presence of a single suitably placed-CO^?₂ group probably cannot explain the binding effect of the-OH group and that additional interactions will be found in the adenosine deaminase active site.