Computer-aided Design of Organophosphorus Inhibitors of Urease

Received Oct 16 th , 2013 Revised April 8 th , 2014 Accepted April 9 th , 2014 Based on the structure of the most potential inhibitor diamidophosphate, various novel groups of inhibitors were developed by knowledge-based design approach with covalent carbon-phosphorus or carbon-phosphorus-carbon bond to improve hydrolytic stability to inhibit the microbial ureases. Designed compounds were evaluated with 10 (LigScore1, LigScore2, PLP1, PLP2, JAIN, PMF, PMF04, LUDI_1, LUDI_2 and LUDI_3) different scoring functions implemented in Discovery Studio and conformation analysis by AutoDock package. Keyword:


INTRODUCTION
Urease (urea amidohydrolase; E.C.3.5.1.5)is an enzyme that holds a salient place in the history of science (Figure 1).Urea was the first organic compound synthesized in laboratory [1], while urease derived from seeds of the Jack bean (JB) plant [2] is the first crystallized [3] and known as nickel containing enzyme [4].It catalyzes urea hydrolysis in the nitrogen mineralization to form ammonia and carbamate, which later decomposes into ammonia and bicarbonate [5] [6].First urease cleaved urea and produce one ammonia molecule and one of carbamate.Carbamate then decomposes in to ammonia and carbonic acid.Then, Carbonic acid equilibrates in water.As two molecules of ammonia become protonated to produce ammonium and hydroxide ions (Scheme 1).As a result there is a rise in the pH of the environment.Ammonia molecules thus formed are protonated by water at physiological pH, whereas the carbonic acid dissociates and cause an increase in pH [5] [6] [7] [8].It involves environmental nitrogen transformations to provide nitrogen source to the organisms like algae, bacteria, fungi and plants [6].But then, the reaction catalyzed by the dinuclear nickel active site of urease causes an aggregation of ammonia and a sharp pH increase, which has negative side effects in agriculture and health.For example, urease serves as a virulence factor in pathogens that are responsible for the development of diseases like kidney stones, pyelonephritis, and peptic ulcers [5] [6].
The precise information for the regions of the enzyme that is involved in the binding of inhibitors or substrates is the base in designing the efficient inhibitors which are capable to complement all the structural requirements for a close interaction.The urease active site (Figure 2) was found to contain pseudooctahedral, paramagnetic and bi-nuclear nickel ions in all the complexes of enzyme inhibitor analyzed so far [9] [10].The information of active site of urease was given through the resolved crystal structures for bacterial ureases from Klebsiella aerogenes [11] [12] and Bacillus pasteurii [9].The active site was displayed to contain a binuclear nickel centre, in which the Ni-Ni distance was found 3.7Å in Bacillus pasteurii.In the center two nickel ions are bridged by a carbamylated lysine through its O-atoms, along Ni(1) further coordinated by two histidines with their N-atoms, and Ni(2) by two histidines also through N-atoms and furthermore by aspartic acid through its Oatom.   [(Color scheme: carbon-grey; nitrogen-blue; oxygen-red; two nickel ions-yellow).Hydrogen bonds are shown as dashed lines.K=lysine.) Along with, the Ni ions are bridged by a hydroxide ion (WB), which along with two terminal water molecules, W1 on Ni(1), W2 on Ni(2), and W3 located near the opening of the active site, which forms an Hbonded water tetrahedral cluster filling the active site cavity.It is the cluster (the carboxylate group of the carbamylated lysine and the hydroxide molecule) that urea replaces when binding to the active site for the reaction [11].As a consequence of above ligations, Ni their coordination geometry is pseudo square pyramidal and pseudo octahedral, respectively.In another consideration, urease can severely decrease the efficiency of urea fertilizers to cause the release of large amounts of ammonia and further induce plant damage by ammonia toxicity and soil pH increase [11].So, to control the rate of the enzymatic urea hydrolysis using urease inhibitors is an important goal.Large quantities of urea produced as a result of biological process.Each human being produces approximately 10 kg of urea per year.Spontaneous degradation of urea occurs with a half life of approximately 3.6 years [1], but in the presence of urease, the hydrolysis of urea is 104 times faster [7].

RESEARCH METHOD
The crystal structure of Bacillus pasteurii urease in complex with inhibitor DAP [13] retrieved from protein data bank [14] (PDB Code: 3UBP) was used as the starting point.To organize the active site of enzyme His137, His139, Ala170, Lys220, His222, His249, His275, Ala279, Gly280, Leu319, Cys322, His323, Ala366, Met367 and Asp363 amino acid residues and two Nickel ions (NI900,NI901) are selected [15] [16].The structure of ligand (DAP) was appropriately modified by using Sketching module and fragment building tools in Discovery Studio 3.1 client program.In structure, missing bond orders, charges and angles were assigned and explicit hydrogens and hydrogen bonds were added.

Molecular modeling
From the created active site of enzyme, both Nickel ions and amino acids which are near and tightly bounded to them (His 137, His139, Lys220, His249, His275, Ala279 and Leu319) were applied to the fixed atom constraint tool of simulation [17] module of Discovery Studio 3.1 client program to limit the energy minimization.CHARMm [18] [19] force field was applied to the whole receptor-ligand complex.Apart from these, other part of active site was subjected to energy minimization using 1000 steps of "smart minimizer", with none of the implicit solvent model and with a dielectric constant of 1.0, Nonbonded List Radius was set to 14 (nonbond higher cutoff distance 12, nonbond lower cutoff distance 10), With fixed minimization constraints and with Electrostatics spherical cutoff until either a RMS gradient of 0.1, or energy change and save result frequency of 0.0, was reached.

Ligand Fit docking and scoring
Ligand Fit, a modern docking program within Discovery Studio (Accelrys, San Diego, USA), was used for all runs.The Receptor-ligand complexes obtained after minimization were further used to determine the ligand binding affinity.The complex was splitted into the enzyme active site part and ligand part.By selecting only the active site part and using "Define and Edit binding site from current selection tool" of "Receptor-ligand interaction" module from the Discovery Studio 3.1 client, binding site sphere (of about 9.9 A°) was defined.After selecting both part (active site part and ligand part), docking studies were subjected to the "score ligand poses" module of Discovery Studio 3.1 client and 10 scoring functions (LigScore1 [20], LigScore2 [20], PLP1 [21], PLP2 [21], JAIN [22], PMF [23], PMF04 [23], LUDI_1, LUDI_2, LUDI_3 [24] [25]) were implemented to evaluate the ligand binding affinity [26] [27] [28].

Molecular docking by AutoDock
Software AutoDock [29] [30] including a graphical user interface, MGL Tools [31] was utilized to generate grids, calculate dock score and assess the conformers.The structure of compounds were drawn using Discovery Studio client 3.1 and the energy minimization was implemented using CHARMm force field [18] [19] with smart minimize method to achieve a local minimum structure.These energy minimized structures are recognized for docking and the correlated pdbqt files were created in AutoDock.AutoDock needs the receptor and ligand coordinates in either Mol2 or PDB format.Other than the water molecules which are present in the active site (HOH 972, HOH 990, HOH 1043, HOH 1046, HOH 1167, HOH 1168 and HOH 1245), all other Nonpolar hydrogens were discarded from the receptor file (PDB [32] code: 2UBP) [9] and their partial charges were calculated to the parallel carbon atoms.
The receptor file was converted into the pdbqt file format containing the receptor atom coordinates, partial charges and solvation parameters.The ligand file was reformed into a pdbqt file and torsions were determined.The grid calculations were implemented and maps were calculated with the use of AutoGrid [33] program.The grid maps were centered on the ligand binding site in the dimension of 120120120 points (x, y, z).The grid spacing was 0.375 Å and other AutoDock parameters were used default for docking.All docking runs were carried out using the Lamarckian genetic algorithm [34] and the achieved dock scores were noted in Kcal/mol.The docking protocol applied in analysis involved of 200 independent runs of each ligand, utilizing an initial population of 250 randomly distributed individuals, a maximum number of 75 × 10 6 energy evaluations, number of generation of 27 × 10 3 , a mutation rate of 0.02, a crossover rate of 0.8 [35].

RESULTS AND ANALYSIS
Knowledge-based method is based on approved technique applied in the drug discovery, established as "scaffold-hopping" [36] [37] where the objective is to design a new structure starting from a known active compound through the modification of the central core of the molecule [38].Generally, basic or initial structure and analogue based design studies are done using Glide, Gold, Ligand Fit or Catalyst to establish a comparative model, then fragment-based and knowledge-based approaches are applied to design molecules of selective inhibitors [39]    BPU is an heteropolymeric molecule (αβγ) 3 with exact threefold symmetry and contains flexible subunit composition which depends on organism.The structure of the active site of urease is highly conserved which contains two nickel ions and has a comparably small volume.Urease inhibitors which are distinct in structure have been effectively identified.The most efficient urease inhibitors are Diamidophosphate (DAP) (Scheme 2) and its derivatives, which hydrolyze to the active molecule (DAP) in the active site [43].
DAP is a transition state analogue.It loses stability in aqueous environments because it contains hydrolyzable (especially at low pH) P-N bonds in its structure [44].So the effort was done to modify the structure of this transition state analogue by using highly stable P-C or C-P-C bonds to improve its activity and stability against Bacillus pasteurii urease.
By using the knowledge-based design approach, 44 different compounds were designed to evaluate their potency against BPU.After designing the novel compounds using the knowledge of already synthesized urease inhibitors, they were energy minimized to the closest local minimum using the molecular mechanics CHARMm force field implemented in Discovery Studio.To study the interaction between the ligand and enzyme active site, all 44 compounds were docked in to the enzyme active site and were evaluated with 10 different scoring functions (Ligscore 1, Ligscore 2, PLP1, PLP2, Jain, PMF, PMF04, Ludi-1, Ludi-2, Ludi-3) of Discovery Studio package.
Automated docking was used to locate the appropriate binding orientations and conformations of different inhibitors in the BPU.To perform the task, genetic algorithm routine implemented in the program AutoDock was employed.Kollman charge, atomic solvation parameters and fragmental volumes were assigned to the protein using MGL Tools package.The program AutoGrid was used to generate the grid maps.Lamarckian genetic algorithm was applied for minimization using default parameters.The standard docking protocol was then applied by using AutoDock software package and binding free energies (G b , Kcal/mol) were obtained.
Inhibitor C-  1) (Scheme 3) are top ranked compounds according to evaluation of 10 scoring functions and binding free energy of AutoDock and these 8 compounds have also obtained highest number of conformations which were quite well overlaid.The inhibitors constructed by using phenyl/aromatic/aliphatic rings with fluorine/chlorine substitutions were obtained top ranked compounds in most of the scoring functions among 44 different structures.By considering all scoring functions, inhibitor C-36 has achieved reliable scores values in all scoring functions (Figure 3).To sum up the attempt of designing organophosphorus compounds as Bacillus pasteurii urease inhibitors, many different compounds containing phosphate, phosphonate, phosphinate, phenyl/ cyclohexyl, pyrrole/cyclopentane in its structure were tested in silico based on knowledge of available inhibitors to improve its potency.From 8 of 10 scoring functions suggest that the compounds containing phenyl/ cyclohexyl/ pyrrole/ cyclopentane can be more potent structures as urease inhibitors than the compounds like phosphates/ phosphonates or phosphinates.

Figure 1 .
Figure 1.Ribbon diagram of the enzyme structure of bacillus pasteurii urease.The blue, light blue and light gray ribbons display α, β and γ subunits, respectively

Figure 3 .
Figure 3. Modeled structure of inhibitor C-36 to active site of Bacillus pasteurii urease.Hydrogen bonds are indicated as green lines.(Discovery Studio): the amino acid residue His222 of enzyme involved in hydrogen bond interaction with phosphinic acid group of inhibitor C-36 with a distance of 1.5 Å.

Table 1 .
Results of scoring functions of selected compounds and their binding energy with the enzyme.