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Protease-inhibitor interactions--a structural insight.

Introduction

Proteolytic enzymes (best termed peptidases) are essential for the survival of all kinds of organisms, and are encoded for by approx. 2% of all genes (Barrett et al., 2001). Despite their life-giving functions, enzymes that break down proteins are potentially very damaging in living systems, so their activities need to be kept strictly under control. Several distinct mechanisms exist for the control of excessive peptidase activity, important amongst which are the interactions of the enzymes with proteins that inhibit them. It is likely that all of the proteins considered have the potential to attenuate the activities of peptidases both in vitro and in vivo by the formation of complexes with the enzymes. Valuable proposals have been made as to how one can assess the physiological relevance of an inhibitor (Turk et al., 2002). The scientific study of the peptidase inhibitors is nearly as old as that of the peptidases themselves. Hundreds of protein inhibitors of peptidases are now known and they are the subjects of thousands of research communications. The research is driven by the many potential applications of knowledge about the inhibitors in medicine, agriculture and biotechnology. At the most fundamental level, an understanding of the mode of interaction of protein inhibitors with enzymes may suggest novel approaches to the design of synthetic inhibitors for use as drugs. Many naturally occurring inhibitors, such as the anticoagulant hirudin, are being used as the basis of engineered proteins for injection in their own right (De Filippis et al., 2002). There are a number of inherited diseases that are attributable to abnormalities in peptidase inhibitors. These include forms of emphysema, epilepsy and hereditary angioneurotic oedema. Netherton syndrome (Lomas et al., 2001, Ritchie, B. C 2003, Lehesjoki, A. E 2003, Bitoun et al., 2002) such diseases may be susceptible to treatment with the inhibitors administered as drugs, with synthetic inhibitors that take over their function, or with the natural inhibitors made available by gene therapy. Excessive proteolytic activities may well contribute to a number of disease conditions and, again, gene therapy to introduce inhibitors is under consideration (Krol et al., 2003 and McKay et al., 2003). In agriculture, genetically modified crop plants expressing inhibitors of the digestive enzymes of their insect pests are already under study (Telang et al, 2003). This active field of research generates a rapid flow of information, but the storage and retrieval of all the new information that is being obtained about the peptidase inhibitors are handicapped by difficulties of nomenclature.

In what was perhaps the most significant review that has been written on the peptidase inhibitors, (Laskowski, M., Jr. and Kato, I. 1980) deplored the confusion of nomenclature that existed in the field in 1980. They pointed out that inhibitors are commonly discovered by their activity against readily available enzymes, most commonly trypsin, chymotrypsin or subtilisin, and then are named after the source organism or tissue, as 'Streptomycin subtilisin inhibitor' or 'pancreatic trypsin inhibitor'. Such names give no clue to the relationships of the inhibitors, and make it difficult to know whether information that is available about the mechanism of action of one inhibitor can correctly be applied to another. It was evident to (Laskowski, M., Jr. and Kato, I.1980) that peptidase inhibitors could best be classified in their homologous families, but the sequence information then available allowed only about a dozen families to be recognized. The names used for peptidase inhibitors have not improved since 1980, but there is now a wealth of sequence data for these proteins, and the time seems right to make a new attempt at a systematic classification of them.

Clans of Inhibitors

A clan of peptidase inhibitors contains all the modern-day inhibitors that have arisen from a single evolutionary origin of inhibitors. It is a group of inhibitors in one or more families that show evidence of their evolutionary relationship by their similar tertiary structures. Each clan of inhibitors has a two-letter identifier, in which the first letter is "I".

[FIGURE 1 OMITTED]

Specificity of serine protease-inhibitor interactions

Trypsin and chymotrypsin are both serine proteases. The two enzymes have high sequence identity and their tertiary structures are very similar. In the chymotrypsin index, His-57, Asp-102, and Ser-195 form the catalytic triad, residues 189-195, 214-220, and 225-228 form the primary substrate-binding pocket called S1 binding pocket (Ma et al, 2005). Residues 185-188 and 221-224 form two loops near the S1 pocket, called L1 and L2, respectively. Catalytic mechanisms of these two proteases are similar, but their substrate specificities are different. Trypsin favors basic residues like lysine and arginine; chymotrypsin favors aromatic residues like phenylalanine, tyrosine, and tryptophan. The S1 binding pocket in trypsin and chymotrypsin are almost identical in primary sequences and backbone tertiary structures. An important difference is that residue 189 is a negatively charged Asp in trypsin and a polar Ser in chymotrypsin. This residue lies at the bottom of the S1 binding pocket and determines different S1 pocket chemical properties. This difference was once used to explain the different specificity of trypsin and chymotrypsin. But the mechanism is not that simple other residues are also involved in the specificity of enzyme-inhibitor interactions (Ma et al., 2005).

Residue Asp-189 plays an important dual role in trypsin; it defines the primary specificity for Arg side chains. This role is shared by other proteases with trypsin-like specificity. Replacement of Asp-189 with Ala, Asn, Glu, and Ser drastically reduces the specificity toward substrates carrying Arg or Lys at P1, whereas it has little or no effect toward the hydrolysis of substrates carrying Phe at P1. These findings confirm the important role of Asp-189 in substrate recognition by trypsin like proteases (Prasad et al., 2004). Specificity of thrombin-like serine proteases is usually categorized in terms of the P1-S1 interaction. The S1 site is a pocket adjacent to Ser-195, formed by residues 189-192, 214-216, and 224-228. Specificity pocket is usually determined by the residues at positions 189, 216, and 226 (Perona, J. J. and Craik, C. S. 1995, Czapinska, H. and Otlewski, J.1999). For example, the specificity of chymotrypsin correlates with the hydrophobicity of the P1 residue, with P1-Phe preferred over Ala (Knowles 1965). The combination of Ser-189, Gly-216, and Gly-226 create a deep hydrophobic pocket in thrombin that accounts for this specificity (Blow, D. M. 1997). Asp-189, Gly-216, and Gly-226 create a negatively charged S1 site that accounts for trypsin's specificity for substrates containing Arg or Lys at P1 (Huber et al., 1974). Elastase prefers substrates with small aliphatic residues at P1; the S1 site of elastase is smaller than the S1 sites of chymotrypsin and trypsin due to the presence of Val-216 and Thr-226 (Shotton, D. M. and Watson, H. C. 1970).

The active site of thrombin is occluded by the B and C insertion loops, which impede docking of macromolecular substrates and inhibitors to the active site pocket. These insertion loops are unique to thrombin. In the crystal structure of thrombin, Tyr60a, Pro60b, Pro60c, and Trp60d of the B-insertion loop form a lid over the S2 specificity pocket of thrombin. There are also several variant residues in the extending binding pockets of thrombin and factor Xa that can influence specificity of these enzymes in their reactions with antithrombin. Residue 192, at the base of the active site pocket, is another variant residue that is known to influence the S3 specificity of thrombin, factor Xa, and other coagulation proteases. The critical role of Glu-192 in restricting the reactivity of thrombin with the serpin, [[alpha].sub.1]-antitrypsin, and the Kunitz inhibitors, bovine pancreatic trypsin inhibitor and tissue factor pathway inhibitor is well studied (Rezaie, A. R. 1998).

A virus (HAV) 3C peptidase was the first structure identified for a viral 3C enzyme that exhibited the three-dimensional fold of the chymotrypsin family of serine peptidases but had a cysteine sulfur atom instead of the serine oxygen as the nucleophile. The structure of HAV 3C was unusual in that the Asp residue expected as the third member of the catalytic triad did not interact with the general base His (James, M. N. 2006). The 3Cpro is distinguished from most other proteases by the fact that it has a cysteine nucleophile but with a chymotrypsin-like serine protease folding. This unique protein structure together with its essential role in viral replication made the 3Cpro an excellent target for antiviral intervention. (Wanga, Q. M. and Chen, S. H. 2007)

Computational methods are needed to exploit the structural information to understand specific molecular recognition events and to elucidate the function of the target macromolecule. This information should ultimately lead to the design of small molecule ligands for the target, which will block/activate its normal function and thereby act as improved drugs.

Materials and Methods

In this work we have focused on the studies of the interactions of proteases with their inhibitors in known structures of the proteases-inhibitors complexes. For this purpose we focused our attention on two well-studied proteases, trypsin and thrombin, with large number of known structures of the complexes. The protein data bank files were downloaded as listed in Table.I for thrombin and in Table. II for trypsin.

Structural superposition of protease along with the inhibitors

All the structures (Table.I) of a selected set of thrombin, containing the inhibitor bound at their active sites, were superposed with a reference structure a structure of thrombin (1A2C) using our in-house software, MODELYN, with respect to all the C[alpha] atoms of the proteases common to both the structures. This process led the superposition of the inhibitor bound to these enzymes allowing us to analyze the common areas of the inhibitors participating in the interaction. Similarly, another set of structures (Table.II) for another well-known protease, trypsin and superposition was done on its x-ray structure (1AQ7).

Identification of the atoms involved in the interactions

Distances of all the atoms of the inhibitor within the interacting distance of 4 [Angstrom] were calculated using MODELYN. This helped us to identify the atoms involved in protease-inhibitors interactions. The nature of chemical forces involved in binding was analyzed using these data.

Analysis of hydrogen-bonding pattern and calculation of interactions energies

Hydrogen bonds between the protease and inhibitors were calculated using InsightII software to identify the atoms involve in hydrogen bonding. Complexes with the modeled structures were predicted by repeated energy minimization and molecular dynamics. The DOCKING module of InsightII was used to calculate the free energy of interactions between the proteases and their inhibitors both in water and protein water environment.

Calculation of free energies of inhibitor binding, [DELTA]G (bind)

The free energy of binding, [DELTA]G (bind), was calculated according to linear interaction energy (LIE) method developed by Aqvist and Samuelsson (1994) and subsequently used for characterization protein ligand interactions (Luzhkov ). The equation is given below:

[DELTA]G (bind) = [alpha][DELTA] [V.sub.1-s.sup,vdW] x +[beta][DELTA] [V.sub.1-s.sup.el]

where [V.sub.1-s.sup.vdW] and [V.sub.1-s.sup.el] denote the Lenard-Jones and electrostatic interactions between the ligand and its surroundings. The symbol, [DELTA], denotes the difference between the energies of the ligand in the protein-water and water environments. The values of [alpha] and [beta] were taken as 0.16 and 0.5 respectively as used for inhibitor binding to the Plasmepsin IV, a protease from Plasmodium falciparum (Luzhkov, et al, 2006). The Lenard-Jones and electrostatic interactions were calculated using the DOCKING module of InsightII.

Results and Discussion

Interaction of Thrombin with its Inhibitors

Common inhibitor binding pockets

In search of the common inhibitor binding pockets of thrombin, sets of experimental structures with bound inhibitors (Table.I) were selected. All the enzyme structures containing the bound inhibitor was superposed with respect to all the corresponding C[alpha] atoms common to both the enzymes of complexes. This led to indirect superposition of all the inhibitors sitting in the specificity pockets of the enzyme. The mutually superposed structures of thrombin showed a deviations (RMSD) ranging from 0.25 to 0.53 [Angstrom]. These deviations are partly due to differences in the 3-D structure determinations in different experimental conditions at different laboratories and partly due to influence of inhibitor binding on the enzyme structures. Hence, such deviations are in a reasonable limit of around 0.5 [Angstrom].

[FIGURE 2 OMITTED]

Fig.2A shows the multiple structural alignments of all the inhibitors involve in enzyme binding. It may be noted that all the inhibitors ensembles in a common space giving the replica of the binding site. Only three enzymes had some groups extended out side the common core of the rest of the inhibitors.

When these inhibitors were taken out of the ensemble the superposed inhibitors occupied a very compact space (Fig.2B), resembling the complementary space of the binding pocket. The composed surfaces of these inhibitors and their electrostatic potentials are shown in Fig.2C & D. The complimentary surfaces show patches of varied electrostatic potentials, some patches of strong positive (blue), negative (red) and neutral segments (white or mixed). This reflects the composite chemical nature of the binding pocket on the protease, thrombin. It is well known that the protease-inhibitor interactions are mediated through a number of sites and sub-sites on the enzyme (Hedstrom, L. 2002). Therefore, we identified all the atoms of the enzyme as well as on the inhibitors, which are in close proximity of the experimentally determined structures of the selected set complexes.

Table.III presents the atoms involved in the interaction of the inhibitors with thrombin in reference to the specificity pockets S1 and S2. The interaction between two atoms, which are in close proximity are said to be polar when both the partners are polar in nature which have either hydrogen bonding, dipole-dipole or charge-charge interactions. The S1 site of thrombin contains Asp-189 as the key specificity determining group; the other two residues in S1 pocket, Gly-216 and Gly-226 (14, 15) are essential for maintaining the conformation around the pocket and providing space to accommodate the complementary groups. Examination of atoms interacting at the S1 site as shown in Table. III reveals the polar nature of the interactions. These findings would help in the process of rational inhibitor design to incorporate groups with proper polarity.

On the other hand, the S2 specificity pocket of thrombin provides the non-polar type of interaction. A non-polar interaction results when at least one of the interacting partners is non-polar. These non-polar interactions are mainly van der Waals type of close atomic contacts or stacking interaction with ring systems containing [pi]-electron clouds. In thrombin the S2 specificity site, the main residues are Tyr-60A, Pro-60B, Pro-60C, Tyr-60D; tyrosine though has a polar hydroxyl group, are very good in staking interactions. Two proline residues serve the duel purpose of providing a highly rigid pocket and supplying polar van der Waals contacts. Atomic level identification of the interacting partners in S2 site as listed in Table.III establishes this non-polar nature of the site for the selected set of thrombin-inhibitor complexes.

Hydrogen-bonding patterns of Thrombin-Inhibitor Interactions

Hydrogen-bonding interactions play major roles in the specificity determination of binding pockets in biological systems, hence, we analyzed the hydrogen-bonding patterns in the binding of thrombin with its inhibitors and shown in Table.IV. It may be noted that all the complexes exhibit a network of hydrogen bonding which include many important specificity pocket residues (shown in bold letters). In rational design of protease inhibitors it would be a good idea to maintain these hydrogen bonds and the aim will also be to incorporate more such interaction wherever possible. Judicial inclusion of newer groups in the designed inhibitors should be placed in such positions so as to maximize these interacting groups.

Empirical Energies of Thrombin-Inhibitor Interactions

The values of energies of interaction provide an important index of binding affinity between the protease and inhibitors. The DOCKING module of InsightII was used to calculate the empirical energy showing the contributions from the van der Waals and electrostatic components of the interactions between thrombin and its inhibitors and presented in Table.V. It is to be noted that all the interaction energies are negative indicating stable complex formation; van der Waals contributions ranged from -76 to -25 Kcals/mole and the electrostatic contributions ranged from -213 to -20 Kcals/mole. It is to be noted that these interaction parameters are the primary guides in the process of inhibitor design. The inhibitors shown in Table.V are those of known inhibitors in the experimental structures of the complexes. The idea is to enhance the binding affinity by decreasing the free energy of interaction. These experimental structures may serve as the starting point in the process of drug design to make better inhibitors followed by improvements in other properties of drugs using the advanced computer-aided techniques. It should be admitted that these energy parameters, though provide initial guidance, there are many more steps in the overall process of drug design. Methods are being constantly improved for the prediction of binding affinity in terms of [DELTA]G and [K.sub.d] values which needs calculations involving aqueous environment (Luzhkov, et al., 2006). In this study we have used these techniques for a few complexes and compared with wet-lab experimental results as presented later in this chapter.

Interaction of Trypsin with its Inhibitors

Common Inhibitor Binding Pocket of Trypsin

Common inhibitor binding pockets of trypsin were also analyzed, as in case of thrombin; sets of experimental structures with bound inhibitors (Table.II) were selected. All the enzyme structures containing the bound inhibitor was superposed with respect to all the corresponding C[alpha] atoms in a common set of enzyme-inhibitor complex. This process led to indirect superposition of all the inhibitors sitting in the specificity pockets of trypsin. The mutually superposed structures of trypsin showed a deviations (RMSD) ranging from 0.25 to 0.56 [Angstrom], values being similar to thrombin complexes. Hence, these deviations are partly due to differences in the 3-D structure determinations in different experimental conditions at different laboratories and partly due to influence of inhibitor binding on the enzyme structures. Thus, such deviations are in a reasonable limit of 0.5 [Angstrom].

Fig.3A shows the multiple structural alignments of all the inhibitors involve in binding to trypsin. In case of the trypsin complexes, many inhibitors are protein inhibitors with extended structures, but at contact point with the enzyme all the inhibitors converged in a common space giving the replica of the binding site. When only the small the inhibitors, which also occupied the common binding pocket were considered, the ensemble of the superposed inhibitors occupied a very compact space (Fig.3B), resembling the complementary space of the binding pocket. The superposed molecules of these inhibitors are shown in space-filling models coloured by atoms (Fig.2 C&D, front and back views). We also identified all the atoms of the enzyme as well as on the inhibitors, which are in close proximity of the experimentally determined structures of the selected set complexes of trypsin with its inhibitors.

[FIGURE 3 OMITTED]

Table. VI presents the atoms involved in the interaction of the inhibitors with trypsin in reference to the specificity pockets S1 and S2. As in case of thrombin, the interactions between atoms in close proximity are thought to be polar when both the partners are polar in nature and have either hydrogen bonding, dipole-dipole or charge-charge interactions. The S1 site of trypsin contains Asp-189 as the key specificity determining group; the other two residues in S1 pocket, Gly-216 and Gly-226 are essential for maintaining the conformation around the pocket and providing space to accommodate the complementary groups, being the identical amino acids as thrombin. Examination of atoms interacting at the S1 site as shown in Table.VI reveals the polar nature of the interactions.

The S2 specificity pocket of trypsin also provides the non-polar type of interactions although the amino acids are different from those of thrombin S2 specificity pocket. In trypsin the main residues are Ser-39, His-40, Phe-41 and Tyr-151 (Ma et al., 2005); the benzene rings of phenylalanine and tyrosine provides the p-electron cloud for non-polar interactions. The interacting partners in S2 site as listed in Table.VI, which establishes this non-polar nature of the site for the selected set of trypsin-inhibitor complexes. However, there are less non-polar interactions in this specificity pocket of trypsin compared to that of thrombin.

Hydrogen-bonding patterns of Trypsin-Inhibitor Interactions

We also analyzed the hydrogen-bonding patterns in the binding of trypsin with its inhibitors and shown in Table.VII. Like those of thrombin, all the complexes exhibited a network of hydrogen bonding which include many important specificity pocket residues (shown in bold letters).

Many protease inhibitors have been developed using proteases as target for rational design (Turk et al., 2005). It would be useful if these hydrogen bonds are maintained and more are incorporated wherever possible. Judicial inclusion of newer groups in the designed inhibitors should be placed in such positions so as to maximize these hydrogen-bonding interactions.

Empirical Energies of Trypsin-Inhibitor Interactions

The index of binding affinity between the protease and inhibitors is very useful in predicting the stronger inhibitors. The calculated empirical energies interim of contributions from the van der Waals and electrostatic components of the interactions between trypsin and its inhibitors presented in Table.VIII. In case of two selected trypsin-inhibitor complexes showed positive values of empirical energies indicating very unstable complex formation. This may be due some imperfection in the structure of these complexes or due to unfavourable charge distribution as the electrical components were positive while van der Waals component had very high negative values (shown in bold in Table.VIII); however, it was not further analyzed. The interaction energies of other complexes were negative indicating stable complex formation with van der Waals contributions ranging from -87 to -28 Kcals/mole of the same order as those of thrombin-inhibitor complexes and the electrostatic contributions ranged from -322 to -14 Kcals/mole. These experimental structures also may serve as the starting point in the process of drug design to make better inhibitors followed by improvements in other drug-like properties.

Empirical binding energies of modeled protease-inhibitor complexes

We predicted the structures of the protease-inhibitor complexes using our predicted models and calculated their interaction energies using the D0CKING module of InsightII. Table.IX presents the values of the energies of modeled complexes along with the calculated values of energies of the experimental structures of complexes (PDB Codes: 1GBI, 1GBD and 1GBM), which were used to predict the structure of these complexes with the same inhibitor bound to them. Structures of three complexes with three different inhibitors from PDB structures were predicted by homology-based method for the protease from P. furiosus of the SA clan. It may be noted that the values of the empirical interaction energies are comparable to those of the experimental structures (Table.IX). Two other threading based models of the proteases from P. falciparum and N. crassa of the SA clan were used to predict the structures of the complexes using the x-ray structure 1GBM with the inhibitor present in the PDB file. In theses cases also the values of calculated energies are comparable to each other. Thus, it may be stated that the modeled structures predicted by us are suitable for design of inhibitors.

Free energies ([DELTA]G) of the protease-inhibitor interactions

Now methods are evolving for the calculation of binding affinities in terms of [DELTA]G and [K.sub.d] values which can be compared with experimental data obtained from wet laboratory experiments using physico-chemical techniques (Mitchell et al., 1996a, Mitchell et al., 1996b). In this study we have used one such technique, linear interaction energy (LIE) method, for calculation of [DELTA]G values of a few complexes and compared them with wet-lab experimental results as well as with results calculated using other method (Table.X). Biomolecular Ligand Energy Evaluation Protocol (BLEEP) is a knowledge-based method that derives potentials of mean force (PMF) by converting the distribution of atom distances between 2.5 [Angstrom] and 8.0 [Angstrom] in a protein-ligand system into pair potential like functions.

Search of protease sequences in the human genome

Current drug discovery efforts are based on rationally identifying chemical compounds that will bind to therapeutic target molecules such as proteins. These efforts are based on the 3D structure of the target molecule and use a variety of computer-based molecular modeling techniques to exploit the 3D structural information. 0f the approximately 400 known human proteases, approximately 14% are under investigation as drug targets. The initial annotation of the approximately 30,000 human proteome set includes approximately 500 proteases. Bioinformatics analysis can now be performed on complete human protease families. New sequences will require evaluation of their function in normal physiology and human disease. Genomic sequence information will have a central role in the validation of protease drug targets (Southan, C. 2001).

The sequences of proteases from different clans, which were used to predict structures were submitted to Genome BLAST and compared with human genome for examining the suitability for use in target based drug design. Proteases from different pathogenic organism like protozoa, fungi and archea were selected for predicting their 3-D structures. Close sequence homology of these sequences with any human protease sequence may make these proteases unsuitable for using as a drug target.

As a positive control of the human genome search for identification of homologous sequences we used some human protease sequences from different clans, which were used in this study. Although the human protease genes are interrupted by introns, the genomic BLAST search, over all 6 reading frames, could identify homologous sequences in the genome. The identities of the portions of the sequence alignments varied from 34% to 100% in a number of human chromosomes like 1, 3, 4, 8, 12, 13, 16, 17, 18 and 19 with high bit score (229) and significant expect values (3 x [10.sup.-58]). Thus, this type search would be able to identify any significant homology with the pathogenic protease target sequences.

We used all the protease sequences of the clans of the lower organisms, which are potentially pathogenic, modeled by us in the genomic BLAST search in the human genome. It was found that in all the cases no significant match was found with any bigger segment (>10AA). The best expect value of 6 x [10.sup.-9] with 38% identity over a stretch of 100 AA was found with Dictyostelium discoideum protease of the SE clan. The next best expect value (6x10-6) with 40% AA identity over a stretch of 50AA with Pyrococcus abyssi protease of the same clan. These matches were due to some homologous segment from certain domain of the proteases, which may exhibit some structural similarity with the human protease. No other protease that we modeled showed any significant match (Expect value > 0.02). Thus, the designed inhibitors of proteases are very less likely to cause any interference with the human protease.

Conclusion

The aim of the work reported here is to develop a structural perspective about the properties that define the specificity of the binding interface between these inhibitors and trypsin-like serine proteases. Most of the serine protease inhibitors from different sources have been found to have considerable medical and industrial importance and they are being extensively studied to obtain an insight into mechanisms for understanding the specificity of inhibition of enzyme catalysis.

In this work, we have analyzed the nature interaction of serine proteases with their inhibitors in atomic details in the experimental structures. Superposition of the structures of the enzymes with respect to each other brought all the inhibitors in the binding sites of the well studied serine proteases, thrombin and trypsin, giving the complementary shapes of the active sites. The surfaces of the indirectly superposed inhibitors were used to assess the nature of the specificity pockets of these enzymes. Individual complexes were used to identify the atoms of the inhibitors which were in close proximity of the enzymes. Thus, it was established that the nature of S1 specificity site was mostly polar and that of S2 specificity site was mostly non-polar of both the serine proteases, thrombin and trypsin.

The calculation of the van der Waals and electrical components the empirical interaction energies showed that in most of the complexes were stabilized by higher electrostatic interactions. All the enzyme-inhibitor complexes were stabilized by an intricate network of hydrogen binding involving the specificity site residues. I calculated the free energy of binding using the linear interaction energy (LIE) method and compared with the reported values calculated using the BLEEP software and determined experimentally, which were in good agreement. Some of our modeled structures of proteases were used to predict the structures enzyme-inhibitor to demonstrate that these structures can be used for rational design of specific inhibitors. Finally it was established that our modeled proteases of pathogenic organisms bear very little homology with the proteases of human genome.

Acknowledgements

This work was carried out and supported by The Indian Institute of Chemical Biology, Kolkata-700032, West Bengal, India.

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Aparna Laskar (1) *, C.N. Mandal (1) and Aniruddha Chatterjee (2) #

(1) Indian Institute of Chemical Biology (CSIR Unit, Govt. Of India), Jadavpur, Kolkata 700032, West Bengal, India

* Correspondence author E-mail: aparnalaskar@gmail.com

(2) M.Sc Biotechnology, School of Biotechnology, Chemical & Biomedical Engineering, VIT University, Vellore-632014, Tamilnadu, India

# Presently at Department of pathology, Dunedin School of Medicine, University of Otago, New Zealand
Table I: The list of experimental structures of the complexes of
thrombin with the inhibitors, which were used for superposition and
analysis of enzyme-inhibitor interactions.

PDB Source Inhibitor
CODE

1A2C Homosapiens Aeruginosin298-A
1A4W Homosapiens 2EP:2-ethylpiperidinekth2-ketothiazole
1A5G Homosapiens BIC:4-amino-4-benzyl-5-oxo-1,6-diazabicyclo
 [4.3.0] nonane-7-carbaldehyde
1AY6 Homosapiens HHO:[1-(hydroxymethyleneamino)-8-hydroxy-octane]
1BA8 Homosapiens Hirugen, CVS1578
1CA8 Homosapiens 3GA:[3-piperidyl-n-guandino-l-alaninal]
1FPC Homosapiens EPI:[4-ethylpiperidine]
1H8D Homosapiens HirudinI, Lepirudin
1H8I Homosapiens HirudinI, LepirudinTys
1LHC Homosapiens DP7[AC(D) phe-pro-boro-arg-OH]
1LHD Homosapiens DI2[AC-(D) phe-pro-borolys-OH]
1LHG Homosapiens DI5[AC-(D) phe-pro-borohomoornithine-OH]
1PPB Homosapiens Chloro Methyl Ketone
1TMT Homosapiens CGP50,856 (synthetic)
1TOM Homosapiens MIN: Methyl-Phe-Pro-Amino-Cyclohexylglycine
1UMA Homosapiens IN2: [N,N-Dimethylcarbamoyl-Alpha Azalysine]
1UVS Homosapiens BM12.1700

Table II: The list of experimental structures of the complexes of
trypsin with the inhibitors, which were used for superposition and
analysis of enzyme-inhibitor interactions.

PDB Source Inhibitor
CODE

1AQ7 Bos taurus Aeruginosin 98-B
1AZ8 Bos taurus BIS-Phenylamidine Inhibitor
1EJM Bos taurus BPI Aprotinin
1JRS Bos taurus Leupeptin
1MTW Bos taurus DX9:(+)-2-[4-[((s)-1-acetimidoyl-3-pyrrodinyl)
 oxy]-3-7-amidino-2-napthyl) propionic acid
1QL8 Bos taurus ZEN: [4-(6-Chloro-Naphthalene-2-sulfonyl)-
1TPP Bos taurus APPA: (p-amidino-phenyl-pyruvate
1TPA Bos taurus BPTI: Bovine Pancreatic Trypsin Inhibitor
1TYN Bos taurus CTA:[cyclotheonamide A ]
1XUF Bos taurus BAZ:BIS(5-amidino-benzimidazolyl)methane zinc
3PTB Bos taurus Benzamidine

Table III: Interactions in S1 and S2 specificity sites of thrombin in
experimental structures of complexes with inhibitors.

PDB Atoms in S1 (AA: D-189, Atoms in S2 (Y-60A,
Code G-216, G-226) specificity P-60B, P-60C, Y-60D)
 site; polar interactions specificity site;
 non-polar interactions

 Enzyme Inhibitor Enzyme Inhibitor

1A4W 189:CG 350:NE2 60A:OE 373:CM1
 189:OD1 350:NE2 60D:CE3 377:C5
 189:OD21 350:NE2 60D:CE3 377:C4
 216:N 350:NE1 60D:CE3 377:N3
 216:CA 350:CZ 60D:CZ2 373:C2
 216:C 350:N 60D:CE2 375:C3
 216:O 350:CG
 226:CA 350:NE2

1AG6 189:CG 372:CZ 60A:CE2 370:C3
 189:OD1 372:CZ 60A:CZ 370:C3
 189:OD2 372:CZ 60A:OE 370:C3
 216:N 372:NE 60D:CZ2 370:C3
 216:CA 372:NE 60D:CZ2 370:N1
 216:C 370:N3 60D:CE2 370:N1
 216:O 370:C6
 226:CA 372:NE1

1AY6 189:CG 7:CZ 60A:CE1 6:CG
 189:CG 7:NE1 60A:CE2 6:CG
 189:OD1 7:NE1 60A:CZ 6:CG
 189:OD2 7:NE1 60A:OE 6:CG
 216:N 7:NE 60D:CZ2 9:C4
 216:CA 7:NE2 60D:CZ2 9:C6
 216:O 5:CB 60D:CZ3 9:C1
 226:CA 7:NE1 60D:CE2 9:C5

1CA8 189:CG 4:NE1 60A:CE2 2:CG
 189:OI1 4:NE2 60A:CZ 2:CG
 189:OI2 4:CZ 60A:OE 2:CG
 216:N 4:CI 60I:CZ2 2:CE
 216:CA 4:C3 60I:CZ2 2:CG
 216:C 4:C3 60I:CE2 2:CE
 216:O 4:C3
 226:CA 4:NE2

1FPC 189:CG 371:NE2 60A:CB 372:C2'
 189:OD1 371:NE2 60A:CG 372:C1'

 189:OD2 371:NE2 60A:CD 372:C2'
 216:N 371:O 60A:CE2 372:C1'
 216:CA 371:NE2 60A:OE 370:C3
 216:C 371:O 60D:CZ2 370:C2
 216:O 371:CB 60D:CZ2 370:C3
 60D:CE2 372:C5

1H8D 216:N 1:C55 60A:CE1 1:C8A
 216:CA 1:C55 60A:CE2 1:C8A
 216:C 1:C55 60A:CZ 1:C8B
 216:O 1:O15 60A:OE 1:C8A
 60D:CZ2 1:C8B
 60D:CZ3 1:C3
 60D:CE2 1:C1
 60D:CE2 1:C2
 60D:CE2 1:C3

1LHG 216:N 400:C3 60A:CE2 400:C8
 216:CA 400:O3 60A:CZ 400:C8
 216:C 400:N3 60A:OE 400:C15
 216:O 400:O3 60D:CZ2 400:C8
 60D:CE2 400:C8

1LHC 189:CG 400:N3 60A:CE1 400:C10
 189:OD1 400:N4 60A:CE2 400:C10
 189:OD2 400:C6 60A:CZ 400:C10
 216:N 400:C5 60A:OE 400:C17
 216:N 400:N2 60D:CZ2 400:C10
 216:CA 400:N3 60D:CE2 400:C10
 216:C 400:N6
 216:O 400:O4
 226:CA 400:N4

1PPB 189:CG 3:CZ 60A:CE2 2:CG
 189:OD1 3:NE2 60A:CZ 2:CG
 189:OD2 3:CZ 60A:OE 1:CE1
 216:N 1:O 60D:CZ2 2:CD
 216:CA 3:NE2 60D:CE2 2:CD
 216:C 1:N
 216:O 1:CB
 226:CA 3:NE1

1TOM 189:CG 1:N1 60A:CE1 1:C11
 189:OD1 1:N1 60A:CE2 1:C11
 189:OD2 1:N1 60A:CZ 1:C11
 216:N 1:C2 60A:OE 1:C11
 216:CA 1:C2 60D:CZ2 1:C12
 216:C 1:N13 60D:CE2 1:O8
 216:O 1:C2
 216:O 1:C16

1TMT 189:CG 3:NE1 60A:CE2 2:CG
 189:OD1 3:NE2 60A:CZ 2:CG
 189:OD2 3:CZ 60A:OE 2:CG
 216:N 3:NE

 216:CA 3:NE2
 216:C 1:N
 216:O 1:CB
 226:CA 3:NE1

1UMA 189:CG 600:N1 60A:CB 500:N1
 189:OD1 600:N1 60A:CG 500:N1
 189:OD2 600:N1 60A:CD1 500:N1
 216:O 600:C2 60A:CD2 500:N1
 60A:CE1 500:C3
 60A:CE2 500:N1
 60A:CZ 500:C2
 60A:OE 500:C5
 60D:CZ3 600:C11
 60D:CE2 600:C11

1UVS 189:CG 11:N31 60A:CD2 11:C39
 189:OD1 11:E31 60A:CE1 11:C38
 189:OD2 11:C30 60A:CE2 11:C38
 216:N 11:O42 60A:CZ 11:C39
 216:CA 11:C30 60A:OE 11:C38
 216:C 11:N20 60A:EE 11:C5
 216:O 11:E20 60D:CZ2 11:C40
 216:E 11:E27 60D:CZ3 11:C40
 60D:CE2 11:C40

Table IV: Analysis of the hydrogen-bonding pattern of the complexes
of thrombin with its selected inhibitors with known experimental
structures in reference to the specificity pockets. Atom name
convention is the same as in protein data bank (PDB). Atoms of
thrombin in the specificity pockets, which are involved in hydrogen
bonding with the inhibitor, are shown in bold letters.

PDB ID HYDROGEN BONDING
(Enzyme-Inhibitor) Enzyme/Inhibitor: Residue: Atom

 DONOR ACCEPTOR

1A5G A5GI:R372:HH11 A5GE:H189:OD1
(A5GE- A5GI) A5GI:R372:HH11 A5GE:H189:OD2
 A5GI:R372:HH21 A5GE:H189:OD2
 A5GI:R370H:H2 A5GE:H192:OE2
 A5GE:H193:HN A5GI:R372:O
 A5GE:H195:HN A5GI:R372:O
 2A5GE:H195:HG A5GI:R372:O
 A5GI:R370H:HN31 A5GE:H216:O
 A5GI:R372:HE A5GE:H219:O
 A5GI:R372:HH22 A5GE:H219:O

1AY6 AY6I:J7:HH11 AY6E:H189:OD1
(AY6E-AY6I) AY6I:J7:HH21 AY6E:H189:OD2
 AY6I:J7:HH12 AY6E:H190:O
 AY6E:H195:HG AY6I:J7:O
 AY6E:H216:HN AY6I:J5H:O
 AY6I:J7:HE AY6E:H219:O
 AY6I:J7:HH22 AY6E:H219:O

1CA8 CA8E:B57:HE2 CA8I:D4H:O
(CA8E-CA8I) CA8I:D4H:HH2 CA8E:B189:OD1
 CA8I:D4H:HN CA8E:B189:OD2
 CA8I:D4H:HH11 CA8E:B214:0
 CA8E:B216: CA8I:D2H:0
 CA8I:D1H:H1S CA8E:B216:O
 CA8I:D2H:HN CA8E:B216:O
 CA8I:D4H:HH12 CA8E:B219:0

1FPC FPCI:R371:HH21 FPCE:H189:OD1
(FPCE-FPCI) FPCI:R371:HH22 FPCE:H189:OD2
 FPCE:H216:HN FPCI:R371:0
 FPCI:R371:HN FPCE:H216:O
 FPCI:R371:HE FPCE:H219:0

1H8D H8DE:H57:HE2 H8DI:K1H:01A
(H8DE-H8DI) H8DI:K1H:H59 H8DE:H216:O
 H8DE:H219:HN H8DI:K1H:015

1LHC LHCE:H57:HE2 LHCI:400H:01
(LHCE-LHCI) LHCI:400H:HN41 LHCE:H189:OD1
 LHCI:400H:HN31 LHCE:H189:0D2
 LHCE:H193:HN LHCI:400H:02
 LHCE:H195:N LHCI:400H:H02
 LHCE:H195:HN LHCI:400H:02
 LHCI:400H:HN1 LHCE:H214
 LHCE:H216:HN LHCI:400H:O4
 LHCI:400H:HN6 LHCE:H216:0
 LHCI:400H:HN32 LHCE:H219:0

1PPB PPBI:I3:HH11 PPBE:H189:OD1
(PPBE-PPBI) PPBI:I3:HH21 PPBE:H189:OD2
 PPBE:H193:HN PPBI:I3:0
 PPBE:H195:HN PPBI:I3:0
 PPBI:I3:HN PPBE:H214:0
 PPBE:H216:HN PPBI:I1:O
 PPBI:I1:HN2 PPBE:H216:O
 PPBI:I3:HH22 PPBE:H219:0

1THM TMTE:H57:HE2 TMTI:I3:0
(THME-THMI) TMTI:I3:HH11 TMTE:H189:OD1
 TMTI:I3:HH21 TMTE:H189:OD2
 TMTE:H193:HN TMTI:I3:0
 TMTE:H195:HN TMTI:I3:0
 TMTI:I3:HN TMTE:H214:0
 TMTE:H216:HN TMTI:I1:0
 TMTI:I1:HN2 TMTE:H216:O
 TMTE:H219:0 TMTI:I3:HH22

1T0M TOMI:1H:H7 TOME:H214:O
 TOME:H216:HN TOMI:1H:O13

Table V: Calculated values of empirical energies of protein-ligand
interactions of the experimental structures of complexes of thrombin
with its inhibitors with the contributions from van der Waals and
electrical components.

PDB ID Empirical energies of protein-ligand
 interactions

 van der Waals Electrical Total

1A4W - 41.20 -118.12 -159.32
1A5G - 76.14 -90.66 -166.81
1AY6 - 53.28 -153.57 -206.86
1CA8 - 57.74 -28.09 -85.83
1FPC - 48.49 -125.16 -173.65
1H8D - 83.76 -28.12 -110.88
1LHC - 49.96 -57.71 -107.68
1LHD - 40.96 -52.71 -93.67
1LHG - 35.23 -50.71 -85.94
1PPB - 36.66 -212.76 -249.43
1T0M - 50.57 -20.13 -70.71
1TMT - 40.76 -189.28 -230.05
1UMA - 30.23 -54.71 -84.94
1UVS - 25.57 -43.04 -68.61

Table.VI: Interactions in S1 and S2 specificity sites of trypsin in
experimental structure of complexes with inhibitors.

PDB Atoms in S1 (D-189, G-216, Atoms in S2 (S-39, H-40,
Code G-226) specificity site; F-41 and Y-151) specificity
 polar interactions site; non-polar interactions

 Enzyme Inhibitor Enzyme Inhibitor

1ANI 189:CG 8:NZ 40:0 10:CD2
 189:0D1 8:NZ 41:CD1 10:0
 189:0D2 8:NZ 41:CD2 10:0
 216:N 6:0 41:CE2 10:0
 216:0 5:C 151:CE2 10:CD2
 226:CA 8:NZ 151:0E 10:CD1

1AQ7 189:CG 1:C71
 189:0D1 1:N73
 189:0D2 1:C71
 216:N 1:N68
 216:CA 1:N68
 226:N 1:N72
 226:CA 1:C71
 226:CA 1:N72

1AZ8 189:CG 1:C20
 189:0D1 1:N4
 189:0D2 1:C20
 216:N 1:03
 216:CA 1:N3
 216:0 1:03
 226:CA 1:N4

1EJM 189:CG 515:CZ 151:0E 534:CG1
 189:0D1 515:NE1
 189:0D2 515:NE2
 216:N 513:C
 216:CE 515:NE2
 216:0 513:CE
 226:CE 515:NE1

1JRS 189:CI B:HH12
 189:CG B:CZ
 189:0D1 B:CZ
 189:0D2 B:CZ
 189:0D2 B:HH22
 216:N B:C
 216:CE B:HH21
 216:C B:0
 216:C B:NH2
 216:C B:HE
 216:0 B:HH21
 216:H B:HE
 226:N I:HH12

1MTW 189:CG 999:C2
 189:0D1 999:N1
 189:0D2 999:N1
 216:N 999:C4
 216:CE 999:N1
 216:C 999:C9
 216:0 999:C18
 226:CE 999:N2

1QL8 189:0D1 999:C2 151:CZ 999:C30
 216:N 999:C3 151:0E 999:C30
 216:CE 999:C3
 216:C 999:C3
 216:0 999:C8

1TAW 189:CG 15:CZ 39:CE2 17:0
 189:CG 15:NE1 39:CZ 19:CI
 189:0D1 15:NE2 39:0E 19:CI
 189:0D2 15:CZ 40:0 17:CG
 216:N 13:0 41:CE 17:0
 216:CE 15:NE2 41:C 17:N
 216:0 13:CE 41:0 16:CE
 226:CE 15:NE1 41:CI 17:0
 151:CD1 17:SD
 151:CE1 17:CE
 151:CE2 17:CE
 151:CZ 17:CE
 151:0E 17:CE

1TPA 189:CG 15:NZ 39:CD2 19:CD1
 189:0D1 15:NZ 39:CE2 19:CD1
 189:0D2 15:NZ 39:CZ 19:N
 216:N 13:0 39:0E 19:0
 216:0 13:C 40:C 17:NE1
 226:CE 15:NZ 40:0 17:NE1
 41:CE 17:0
 41:C 17:N
 41:0 16:CE
 151:CE 17:CG
 151:CZ 17:CG
 151:0E 17:CG

1TYN 189:CG 246:C46 39:0E 246:C37
 189:0D1 246:N48 41:0 246:C34
 189:0D2 246:N48 41:C 246:C39
 216:N 246:N45
 216:CE 246:N48
 216:0 246:C52
 226:CE 246:N47

1XUF 189:CG 246:N1 41:CE 246:E2'
 189:0D1 246:C7 41:C 246:E2'
 189:0D2 246:EN2 41:0 246:C2'
 216:N 246:C6 41:CB 246:N2'
 216:CE 246:N1 41:CB 246:E2'
 216:C 246:EN1
 216:E 246:C6
 226:N 246:EN2

Table VII: Analysis of the hydrogen-bonding pattern of the complexes
of trypsin with its selected inhibitors with known experimental
structures in reference to the specificity pockets. Atom name
convention is the same as in protein data bank (PDB). Atoms of
trypsin in the specificity pockets, which are involved in hydrogen-
bonding with the inhibitor, are shown in bold letters.

PDB ID HYDROGEN BONDING
(Enzyme-Inhibitor) Enzyme/Inhibitor: Residue: Atom

 DONOR ACCEPTOR

1ANI ANII:I10:HN ANIE:E41:0
(ANIE-ANII) ANII:I8:HZ3 ANIE:E190:O
 ANIE:E192:HE21 ANII:I7:0
 ANIE:E193:HN ANII:I8:0
 ANIE:E195:HN ANII:I8:0
 ANII:I8:HN ANIE:E214:O
 ANIE:E216:HN ANII:I6:0
 ANII:I6:HN ANIE:E216:O
 ANIE:E219:HN ANII:I4:0

1AQ7 AQ7B:1H:H721 AQ7A:189:0D1
(AQ7E-AQ7I) AQ7B:1H:H731 AQ7A:189:0D2
 AQ7A:192:HE21 AQ7B:1H:056
 AQ7B:1H:H57 AQ7A:214:O
 AQ7B:1H:H031 AQ7A:216:N
 AQ7A:216:HN AQ7B:1H:O31
 AQ7B:1H:H18 AQ7A:216:O
 AQ7A:219:HN AQ7B:1H:016
 AQ7B:1H:H68 AQ7A:219:0
 AQ7B:1H:H732 AQ7A:219:0

1AZ8 AZ8I:1H:H35 AZ8E:146:0
(AZ8E-AZ8I) AZ8I:1H:H32 AZ8E:148:0
 AZ8I:1H:H34 AZ8E:148:0
 AZ8E:149:HG1 AZ8I:1H:N2
 AZ8I:1H:H20 AZ8E:189:0D1
 AZ8I:1H:H18 AZ8E:189:0D2
 AZ8I:1H:H21 AZ8E:190:OG
 AZ8E:216:HN AZ8I:1H:O3
 AZ8I:1H:H19 AZ8E:219:0

1EJM EJME:B519:HN EJMI:A39:OH
(EJME-EJMI) EJME:B517:HE EJMI:A40:O
 EJME:B517:HN EJMI:A41:O
 EJMEI:A57:ND1 EJMI:B514:SG
 EJMEI:B539:HE EJMI:A97:0
 EJMEI:B539:HH22 EJMI:A97:0
 EJMEI:B515:HH11 EJMI:A189:0D1
 EJME:B515:HH21 EJMI:A189:0D1
 EJME:B515:HH11 EJMI:A189:0D2
 EJME:B515:HH12 EJMI:A190:OG
 EJME:A192:HE21 EJMI:B514:0
 EJME:A193:HN EJMI:B515:0
 EJME:A195:HN EJMI:B515:0
 EJME:A195:HG EJMI:B515:0
 EJME:B515:HN EJMI:A214:O
 EJME:A216:HN EJMI:B513:O
 EJME:B515:HH22 EJMI:A219:0

1JRS JRSE:A57:HE2 JRSI:B3:0
(JRSE-JRSI) JRSI:B3:HH11 JRSE:A189:0D1
 JRSI:B3:HH22 JRSE:A189:0D2
 JRSI:B3:HH12 JRSE:A190:OG
 JRSE:A192:HE21 JRSI:B2:0
 JRSI:B3:HN JRSE:A214:O
 JRSE:A216:HN JRSI:B1:O
 JRSI:B3:HH21 JRSE:A219:0

1MTW MTWI:999H:N28 MTWE:48:0
(MTWE-MTWI) MTWI:999H:N28 MTWE:48:0D1
 MTWI:999H:H322 MTWE:49:N
 MTWI:999H:H321 MTWE:49:0
 MTWE:51:HN MTWI:999H:N28
 MTWI:999H:023 MTWE:242:0
 MTWI:999H:023 MTWE:245:0

1QL8 QL8I:A600H:H4 QL8E:A57:NE2
(QL8E-QL8I) QL8E:A57:HE2 QL8I:A600H:01
 QL8E:A57:HE2 QL8I:A600H:04
 QL8I:A999H:H1 QL8E:A190:OG
 QL8E:A193:HN QL8I:A600H:02

1TYN TYNE:57:HE2 TYNI:246H:040
(TYNE-TYNI) TYNI:246H:H471 TYNE:189:0D1
 TYNI:246H:H481 TYNE:189:0D2
 TYNI:246H:H472 TYNE:190:OG
 TYNE:192:HE21 TYNI:246H:049
 TYNE:193:HN TYNI:246H:041
 TYNE:195:HN TYNI:246H:041
 TYNI:246H:H12 TYNE:214:O
 TYNI:216:HN TYNI:246H:O50
 TYNI:246H:H51 TYNE:216:O
 TYNI:246H:H482 TYNE:219:0

1XUK XUKE:246H:H11 XUKE:189:0D2
(XUKE-XUKI) XUKE:190:HG XUKI:246H:N2
 XUKE:246H:H12 XUKI:219:0

Table VIII: Calculated values of empirical energies of protein-
ligand interactions of the experimental structures of complexes of
trypsin with its inhibitors with the van der Waals and electrical
contributions.

PDB ID Empirical energies of protein-ligand interactions

 van der Waals Electrical Total

1ANI - 83.91 - 67.51 -151.42
1AQ7 - 53.86 - 42.43 -96.29
1AZ8 - 38.40 - 18.87 - 57.27
1EJM - 95.59 125.63 30.04
1JRS - 35.46 - 82.59 -118.06
1MTW - 43.03 - 16.45 -59.48
1QL8 - 46.24 - 18.20 -64.45
1TAW - 87.56 - 322.81 - 410.37
1TPA - 88.11 101.95 13.83
1TYN - 57.21 - 31.47 - 88.68
1XUF - 28.11 - 14.34 - 42.45

Table IX: Calculated values of empirical energies of protein-ligand
interactions of the modeled structures of complexes in comparison to
the reference experimental structures with the van der Waals and
electrical contributions.

PDB Structure of Empirical energies of protein-ligand
Code Complex interactions

 van der Waals Electrical Total

1GBI x-ray -34.35 -77.12 -111.47
1GBD x-ray -32.38 -90.71 -123.29
1GBM x-ray -41.34 -90.48 -131.82
1GBI Model (P. furiosus) -28.78 -123.80 -152.59
1GBD Model (P. furiosus) -27.91 -43.96 -71.87
1GBM Model (P. furiosus) -22.47 -24.42 -46.90
1GBM Model (P.falciparum) -13.09 -30.63 -43.72
1GBM Model (N. crassa) -41.24 -100.93 -142.18

Table X: Binding free energies ([DELTA]G) calculated by linear
interaction energy (LIE) method and comparison with the values
available in website http://www.mitchell.ch.cam.ac.
uk/pld/background_energy_bleep.html calculated by BLEEP using
potentials of mean force (PMF) method and determined experimental
techniques.

PDB Inhibitors Values of [DELTA]G in (in Kcals/mol)
Code
 Calculated by us BLEEP Experimental*
 using LIE **

 vdW Elec. Total

1ARC TLCK -4.22 -6.38 -3.86
1BCR antipain -5.00 -4.67 -1.53
1BCS chymostatin -7.87 -12.89 -7.70
1BRA Benzamidine -7.70 -8.08 -5.27 -2.49 -2.49
1D3D Hirugen -2.14 -7.68 -4.18 -12.38 -12.38
1DWC argatroban -2.80 -3.40 -2.15 -10.09 -10.09
1EQ9 PMSF -6.39 -4.22 -4.11
1PPH NAPAP -9.61 -7.12 -5.06 -8.48 -8.48
1TNG 2-amino-methyl- -6.42 -3.70 -2.87 -4.00 -4.00
 cyclohexane
1TNH 2,4-fluorobenzy- -5.96 -6.62 -2.09 -4.59 -4.59
 lamine
1TNK 2,3-phenylpropyl- -8.94 -2.16 -2.51 -2.03 -2.03
 amine
1KZD tyrostatin -5.25 -14.65 -8.16
1VGC L-para-chloro-1- -5.13 -7.24 -4.42
 acetamido boronic
 acid
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Author:Laskar, Aparna; Mandal, C.N.; Chatterjee, Aniruddha
Publication:International Journal of Biotechnology & Biochemistry
Date:May 1, 2010
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