version date: 1 December 2006 implicitly enclose hydrophobic and solvation/desolvation effects, directly related to the entropic contribution involved in molecular associations. The formation of a complex between a protein and a ligand in aqueous solution can be represented by the following equilibrium 2a+La已PLa where P is the protein, L the ligand, P'l' the new complex, and k+1 and k-l are, respectively, the association and dissociation constants Both Ka(association)and Ka(dissociation) are related to the activity of the reacting species n, but, if extremely dilute solutions are considered, activities can be substituted by concentrations. Starting from the constant values it is possible to calculate the free energy of binding associated to the binding event, using the following relation △G°=- RTIn Kd T is the absolute temperature, R the gas constant and Ago the binding free energy variation measured in standard condition(298%K, I atm, and I M concentration for Po/w is also an equilibrium constant for solute transfer between octanol and water log Po/w=-△AG/2.303RT where r and t are constants It derives that log pol=k△G° where k =-0733 kcal mol-I at 298 K Because ∑a;= log pol is obvious the relationship between hydrophobic atomic constants ai and AG, thus, including both enthalpic and entropic contribution [ 9] HINT can be defined as a natural and intuitive force field, able to estimate, using experimentally determined log P values, not only the enthalpic but also the entropic effects included in noncovalent interactions, like hydrogen bonding, Coulombic forces, acid-base and hydrophobic contact Hydrophobic and polar contacts, both identified as hydropathic interactions, are strictly related to solvent partitioning phenomena. In fact, the solubilization of a ligand in a mixed solvent system, <www.iupac.org/publications/cd/medicinal_chemistry/>
6 implicitly enclose hydrophobic and solvation/desolvation effects, directly related to the entropic contribution involved in molecular associations. The formation of a complex between a protein and a ligand in aqueous solution can be represented by the following equilibrium: Paq. + Laq. ⇄ P'L'aq.' where P is the protein, L the ligand, P'L' the new complex, and k+1 and k–1 are, respectively, the association and dissociation constants. Ka = Kd –1 = [ ] [ ][ ] P L PL Both Ka (association) and Kd (dissociation) are related to the activity of the reacting species n, but, if extremely dilute solutions are considered, activities can be substituted by concentrations. Starting from the constant values it is possible to calculate the free energy of binding associated to the binding event, using the following relation: ∆G° = –RT ln Kd T is the absolute temperature, R the gas constant and ∆G° the binding free energy variation measured in standard condition (298 °K, 1 atm, and 1 M concentration for both reagents and products). Po/w is also an equilibrium constant for solute transfer between octanol and water: log Po/w = –∆G°/2.303 RT where R and T are constants. It derives that log Po/w = k ∆G° where k ≈ –0.733 kcal mol–1 at 298 K. Because Σai = log Po/w it is obvious the relationship between hydrophobic atomic constants ai and ∆G°, thus, including both enthalpic and entropic contribution [9]. HINT can be defined as a natural and intuitive force field, able to estimate, using experimentally determined log P values, not only the enthalpic but also the entropic effects included in noncovalent interactions, like hydrogen bonding, Coulombic forces, acid-base and hydrophobic contacts. Hydrophobic and polar contacts, both identified as hydropathic interactions, are strictly related to solvent partitioning phenomena. In fact, the solubilization of a ligand in a mixed solvent system, k+1 k-1 <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 like water and octanol, involves the same processes and atom-atom interactions as biomolecular interactions within or between proteins and ligands [24]. The program was designed to consider and investigate hydrophobicity and hydropathic interactions in several biological areas. HINT is able to (i calculate hydrophobic atomic constant for each atom in small molecule or even in macromolecule and quantitatively score molecular interactions, (ii) create hydrophobic maps or fields for small molecules in protein environments, (iii) map the hydrophobic and polar nature of the surrounding receptor from the structure of small interacting molecules, providing a hydrophobic interaction template for the definition of secondary and tertiary protein structure, and (iv)suggest modes of inter-helix interactions in trans-membrane ion channel [25]. All these features and capabilities make hinT a suitable tool, not only for the study of single and simple interactions, but also for the virtual screening of organic libraries and for structure-based drug design interactions between atom-atom couples are calculated using the following equation bj=ai Sia s Ti ri+ry where bi represents the interaction score between atoms i and j, a is the hydrophobic atomic constant, S is the SASA, Ti is a logic function assuming-l or +l value, depending on the character of the interacting polar atoms, while Ri and ri are a function of the distance between atoms i and The whole interaction between two molecules, like protein and ligand, or protein and DNA, can be represented as ΣΣb=Ea1 Siai si tii r+r bj>0 identifies favorable interactions, while bi<0 the unfavorable ones. Interactions can be divided into: polar-polar, hydrophobic-hydrophobic, and hydrophobic-polar. While hydrophobic hydrophobic contacts are always positively scored, polar interactions, depending on the charge of interacting groups can be favorable(acid-base), or unfavorable (acid-acid and base-base) Hydrophobic-polar contacts are constantly negatively scored by HINT, so they negatively contribute to the global binding energy. The Hint hydrophobic-polar interaction score term represents an empirical free-energy evaluation for the energy cost to desolate the polar regions of proteins or ligands, placing them in a hydrophobic environment The HINT software allows us to reduce the information from bulk molecule solvent partitioning, to discrete interactions between biological molecules, i.e., ligand-protein, protein-protein, protein- DNA, and protein-ligand-water Small differences have been revealed in hydrophobicity estimations between HINT and CLOGP Some examples are reported in Table 1 [25] <www.iupac.org/publications/cd/medicinal_chemistry/>
7 like water and octanol, involves the same processes and atom–atom interactions as biomolecular interactions within or between proteins and ligands [24]. The program was designed to consider and investigate hydrophobicity and hydropathic interactions in several biological areas. HINT is able to (i) calculate hydrophobic atomic constant for each atom in small molecule or even in macromolecule and quantitatively score molecular interactions, (ii) create hydrophobic maps or fields for small molecules in protein environments, (iii) map the hydrophobic and polar nature of the surrounding receptor from the structure of small interacting molecules, providing a hydrophobic interaction template for the definition of secondary and tertiary protein structure, and (iv) suggest modes of inter-helix interactions in trans-membrane ion channel [25]. All these features and capabilities make HINT a suitable tool, not only for the study of single and simple interactions, but also for the virtual screening of organic libraries and for structure-based drug design. Interactions between atom–atom couples are calculated using the following equation: bij = ai Si aj Sj Tij Rij + rij where bij represents the interaction score between atoms i and j, a is the hydrophobic atomic constant, S is the SASA, Tij is a logic function assuming –1 or +1 value, depending on the character of the interacting polar atoms, while Rij and rij are a function of the distance between atoms i and j. The whole interaction between two molecules, like protein and ligand, or protein and DNA, can be represented as ΣΣ bij = ΣΣ ai Si aj Sj Tij Rij + rij bij > 0 identifies favorable interactions, while bij < 0 the unfavorable ones. Interactions can be divided into: polar–polar, hydrophobic–hydrophobic, and hydrophobic–polar. While hydrophobic– hydrophobic contacts are always positively scored, polar interactions, depending on the charge of interacting groups can be favorable (acid–base), or unfavorable (acid–acid and base–base). Hydrophobic–polar contacts are constantly negatively scored by HINT, so they negatively contribute to the global binding energy. The HINT hydrophobic–polar interaction score term represents an empirical free-energy evaluation for the energy cost to desolvate the polar regions of proteins or ligands, placing them in a hydrophobic environment. The HINT software allows us to reduce the information from bulk molecule solvent partitioning, to discrete interactions between biological molecules, i.e., ligand–protein, protein–protein, protein– DNA, and protein–ligand–water. Small differences have been revealed in hydrophobicity estimations between HINT and CLOGP. Some examples are reported in Table 1 [25]. <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 Table 1 Compound HINT CLOG-P anthrace 4.45 4.49 1.3-butadiene n-butylamine 0.97 hexachlorobenzene 5.79 642 N-nitrosomorpholine 0.55 0.14 cortisone 0.49 testosterone 3.35 HINT PRACTICAL APPLICATIONS 1. PROTEIN-LIGAND INTERACTIONS Within an homogeneous biological set, HINT can be easily used to score and predict the free energy associated to protein-ligand complex formation. Starting from good crystallographic data and well experimentally determined Ki or ICso values (Table 2), it is possible to obtain linear relationships between experimental AG and computationally calculated HINT score values Table 2 reports the HiNT score protein-ligand values calculated for two different homogenous set, formed, respectively, by eight bovine trypsin-ligand complexes and by nine tryptophan synthase-ligand complexes, for which experimental inhibition constants are reported in literature Table 2 PDB code △G° bindin(kca/mo) Hint score ITNJ ITNI ITNG 3PTB PPH bovine trypsin 804 2663 CX9 2TRS 720 tryptophan synthethase tryptophan synthase 2TSY tryptophan synthethase <www.iupac.org/publications/cd/medicinalchemistry/>
8 Table 1 Compound HINT CLOG-P anthracene 4.45 4.49 1,3-butadiene 1.76 1.90 n-butylamine 0.97 0.92 cyclopentane 2.94 2.80 hexachlorobenzene 5.79 6.42 N-nitrosomorpholine –0.41 –0.64 aldosterone 0.55 –0.14 cortisone 0.49 0.20 testosterone 3.35 3.35 HINT PRACTICAL APPLICATIONS 1. PROTEIN–LIGAND INTERACTIONS Within an homogeneous biological set, HINT can be easily used to score and predict the free energy associated to protein-ligand complex formation. Starting from good crystallographic data and well experimentally determined Ki or IC50 values (Table 2), it is possible to obtain linear relationships between experimental ∆G° and computationally calculated HINT score values. Table 2 reports the HINT score protein-ligand values calculated for two different homogenous set, formed, respectively, by eight bovine trypsin-ligand complexes and by nine tryptophan synthase-ligand complexes, for which experimental inhibition constants are reported in literature. Table 2 PDB code protein ∆G°binding (kcal/mol) Hint score 1TNJ bovine trypsin –2.66 677 1TNK bovine trypsin –2.02 720 1TNI bovine trypsin –2.30 834 1TNL bovine trypsin –2.54 1360 1TNG bovine trypsin –3.98 923 1TNH bovine trypsin –4.57 972 3PTB bovine trypsin –6.43 1634 1PPH bovine trypsin –8.04 2663 1CX9 tryptophan syntethase –9.58 2595 1C29 tryptophan syntethase –9.00 2793 1C9D tryptophan syntethase –8.97 3094 1CW2 tryptophan syntethase –8.76 3094 1C8V tryptophan syntethase –8.92 2571 2TRS tryptophan syntethase –7.20 2646 1QOP tryptophan syntethase –7.20 2721 1A50 tryptophan syntethase –8.56 2914 2TSY tryptophan syntethase –4.65 905 <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 10 2000 4000 Hint Score Fig. 2 Plots of experimental AG vS. HINT score units for bovine trypsin(cyan triangle)and tryptophane synthase(green triangle) The regression analyses of bovine trypsin and tryptophan synthase data series are shown in Fig. 2 and, respectively, represented by the following equations AG°=-0.0019HSpL-3.1210 △G°=-00028HSpL-0.5880 with R=0.83,(standard error) SE=0.90 kcal/mol for trypsin-ligand complexes andR=0.87 and SE=1.16 kcal/mol for tryptophan synthase-ligand complexes Thus, it is possible to predict the binding free energy of new hypothetical trypsin or tryptophan synthase ligands, for which the experimental inhibition constant value has not been yet determined just calculating the hinT score value for the new potential complex, as shown in Fig 3 <www.iupac.org/publications/cd/medicinalchemistry/>
9 Fig. 2 Plots of experimental ∆G° vs. HINT score units for bovine trypsin (cyan triangle) and tryptophane synthase (green triangle). The regression analyses of bovine trypsin and tryptophan synthase data series are shown in Fig. 2 and, respectively, represented by the following equations: ∆G° = –0.0019 HSP–L –3.1210 ∆G° = –0.0028 HSP–L –0.5880 with R = 0.83, (standard error) SE = 0.90 kcal/mol for trypsin-ligand complexes and R = 0.87 and SE = 1.16 kcal/mol for tryptophan synthase-ligand complexes. Thus, it is possible to predict the binding free energy of new hypothetical trypsin or tryptophan synthase ligands, for which the experimental inhibition constant value has not been yet determined, just calculating the HINT score value for the new potential complex, as shown in Fig. 3. Hint Score 0 1000 2000 3000 4000 ∆G° (kcal/mol) -2 -4 -6 -8 -10 <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 Predicted △ binding free energy 3000 4000 Hint Score Fig 3 Prediction of the binding free energy of a new potential bovine typsin ligand, from the protein-ligand HINT score It is more difficult to find a good relationship between experimental and computational data, for a heterogeneous set of protein-ligand complexes, characterized by different active site polarity, igands with diverse chemical nature, and inhibition constants varying among 10 or more orderd of magnitude [13]. In the following analysis, 93 different crystallographic protein-ligand complexes were examined and scored, in order to define a general relationship between AG and Hint score Experimental and calculated data, with both the protein nature and the crystallographic resolution values, are reported in Table 3, while the general relation is shown in Fig. 4 Table 3 PDB code Protein Crystal resolution(A) AG binding (kcal/ mol) Hint score IETT bovine thrombin 2.50 2131 IETR bovine thromb IUVT 1A2C human thrombin 8.97 3019 LOw human human thrombi ICu 1C4V human thrombin 10 human thrombin 2.07 IKTT human thrombin 3586 IOYT humaww Upac. org/publications/cd/medicinal_chemistiy9s
10 Fig. 3 Prediction of the binding free energy of a new potential bovine typsin ligand, from the protein–ligand HINT score It is more difficult to find a good relationship between experimental and computational data, for a heterogeneous set of protein–ligand complexes, characterized by different active site polarity, ligands with diverse chemical nature, and inhibition constants varying among 10 or more orderd of magnitude [13]. In the following analysis, 93 different crystallographic protein–ligand complexes were examined and scored, in order to define a general relationship between ∆G° and HINT score. Experimental and calculated data, with both the protein nature and the crystallographic resolution values, are reported in Table 3, while the general relation is shown in Fig. 4. Table 3 PDB code Protein Crystal resolution (Å) ∆G°binding (kcal/mol) Hint score 1ETS bovine thrombin 2.30 –11.17 3623 1ETT bovine thrombin 2.50 –8.00 2131 1ETR bovine thrombin 2.20 –10.49 2848 1UVT bovine thrombin 2.50 –10.38 1834 1A2C human thrombin 2.10 –8.97 3019 1A4W human thrombin 1.80 –8.05 3110 1BHX human thrombin 2.30 –9.30 2283 1D6W human thrombin 2.00 –8.10 4005 1FPC human thrombin 2.30 –9.52 2299 1C4U human thrombin 2.10 –14.09 3882 1C4V human thrombin 2.10 –14.67 4390 1C5N human thrombin 1.50 –6.39 2334 1C50 human thrombin 1.90 –4.75 2498 1D4P human thrombin 2.07 –8.57 3363 1KTT human thrombin 2.10 –8.33 3586 1OYT human thrombin 1.67 –9.85 3660 Hint Score 0 1000 2000 3000 4000 ∆G° (kcal/mol) -2 -4 -6 -8 -10 Predicted binding free energy new ligand <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006