Theoretical Evaluation of Novel Thermolysin Inhibitors from Bacillus thermoproteolyticus. Possible Antibacterial Agents.

The search for new antibacterial agents that could decrease bacterial resistance is a subject in continuous development. Gram-negative and Gram-positive bacteria possess a group of metalloproteins belonging to the MEROPS peptidase (M4) family, which is the main virulence factor of these bacteria. In this work, we used the previous results of a computational biochemistry protocol of a series of ligands designed in silico using thermolysin as a model for the search of antihypertensive agents. Here, thermolysin from Bacillus thermoproteolyticus, a metalloprotein of the M4 family, was used to determine the most promising candidate as an antibacterial agent. Our results from docking, molecular dynamics simulation, molecular mechanics Poisson-Boltzmann (MM-PBSA) method, ligand efficiency, and ADME-Tox properties (Absorption, Distribution, Metabolism, Excretion, and Toxicity) indicate that the designed ligands were adequately oriented in the thermolysin active site. The Lig783, Lig2177, and Lig3444 compounds showed the best dynamic behavior; however, from the ADME-Tox calculated properties, Lig783 was selected as the unique antibacterial agent candidate amongst the designed ligands.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Paying the Price of Desolvation in Solvent-Exposed Protein Pockets: Impact of Distal Solubilizing Groups on Affinity and Binding Thermodynamics in a Series of Thermolysin Inhibitors.

In lead optimization, open, solvent-exposed protein pockets are often disregarded as prospective binding sites. Because of bulk-solvent proximity, researchers are instead enticed to attach charged polar groups at inhibitor scaffolds to improve solubility and pharmacokinetic properties. It is rarely considered that solvent effects from water reorganization in the first hydration shell of protein-ligand complexes can have a significant impact on binding. We investigate the thermodynamic fingerprint of thermolysin inhibitors featuring terminal charged ammonium groups that are gradually pulled from a distal, solvent-exposed position into the flat, bowl-shaped S2' pocket. Even for the most remote attachment, costs for partial desolvation of the polar group next to the protein-solvent interface are difficult to compensate by interactions with the protein or surrounding water molecules. Through direct comparison with hydrophobic analogues, a significant 180-fold affinity loss was recorded, which questions popular strategies to attach polar ligand-solubilizing groups at the exposed terminus of substituents accommodated in flat open pockets.

Elucidating the Origin of Long Residence Time Binding for Inhibitors of the Metalloprotease Thermolysin.

Kinetic parameters of protein-ligand interactions are progressively acknowledged as valuable information for rational drug discovery. However, a targeted optimization of binding kinetics is not easy to achieve, and further systematic studies are necessary to increase the understanding about molecular mechanisms involved. We determined association and dissociation rate constants for 17 inhibitors of the metalloprotease thermolysin by surface plasmon resonance spectroscopy and correlated kinetic data with high-resolution crystal structures in complex with the protein. From the structure-kinetics relationship, we conclude that the strength of interaction with Asn112 correlates with the rate-limiting step of dissociation. This residue is located at the beginning of a ß-strand motif that lines the binding cleft and is commonly believed to align a substrate for catalysis. A reduced mobility of the Asn112 side chain owing to an enhanced engagement in charge-assisted hydrogen bonds prevents the conformational adjustment associated with ligand release and transformation of the enzyme to its open state. This hypothesis is supported by kinetic data of ZFPLA, a known pseudopeptidic inhibitor of thermolysin, which blocks the conformational transition of Asn112. Interference with this retrograde induced-fit mechanism results in variation of the residence time of thermolysin inhibitors by a factor of 74 000. The high conservation of this structural motif within the M4 and M13 metalloprotease families underpins the importance of this feature and has significant implications for drug discovery.

N-tuple topological/geometric cutoffs for 3D N-linear algebraic molecular codifications: variability, linear independence and QSAR analysis.

Novel N-tuple topological/geometric cutoffs to consider specific inter-atomic relations in the QuBiLS-MIDAS framework are introduced in this manuscript. These molecular cutoffs permit the taking into account of relations between more than two atoms by using (dis-)similarity multi-metrics and the concepts related with topological and Euclidean-geometric distances. To this end, the kth two-, three- and four-tuple topological and geometric neighbourhood quotient (NQ) total (or local-fragment) spatial-(dis)similarity matrices are defined, to represent 3D information corresponding to the relations between two, three and four atoms of the molecular structures that satisfy certain cutoff criteria. First, an analysis of a diverse chemical space for the most common values of topological/Euclidean-geometric distances, bond/dihedral angles, triangle/quadrilateral perimeters, triangle area and volume was performed in order to determine the intervals to take into account in the cutoff procedures. A variability analysis based on Shannon's entropy reveals that better distribution patterns are attained with the descriptors based on the cutoffs proposed (QuBiLS-MIDAS NQ-MDs) with regard to the results obtained when all inter-atomic relations are considered (QuBiLS-MIDAS KA-MDs - 'Keep All'). A principal component analysis shows that the novel molecular cutoffs codify chemical information captured by the respective QuBiLS-MIDAS KA-MDs, as well as information not captured by the latter. Lastly, a QSAR study to obtain deeper knowledge of the contribution of the proposed methods was carried out, using four molecular datasets (steroids (STER), angiotensin converting enzyme (ACE), thermolysin inhibitors (THER) and thrombin inhibitors (THR)) widely used as benchmarks in the evaluation of several methodologies. One to four variable QSAR models based on multiple linear regression were developed for each compound dataset following the original division into training and test sets. The results obtained reveal that the novel cutoff procedures yield superior performances relative to those of the QuBiLS-MIDAS KA-MDs in the prediction of the biological activities considered. From the results achieved, it can be suggested that the proposed N-tuple topological/geometric cutoffs constitute a relevant criteria for generating MDs codifying particular atomic relations, ultimately useful in enhancing the modelling capacity of the QuBiLS-MIDAS 3D-MDs.

Synthesis, experimental evaluation and molecular modelling of hydroxamate derivatives as zinc metalloproteinase inhibitors.

Enzymes of the M4 family of zinc-metalloproteinases are virulence factors secreted from gram-positive or gram-negative bacteria, and putative drug targets in the treatment of bacterial infections. In order to have a therapeutic value such inhibitors should not interfere with endogenous zinc-metalloproteinases. In the present study we have synthesised a series of hydroxamate derivatives and validated the compounds as inhibitors of the M4 enzymes thermolysin and pseudolysin, and the endogenous metalloproteinases ADAM-17, MMP-2 and MMP-9 using experimental binding studies and molecular modelling. In general, the compounds are stronger inhibitors of the MMPs than of the M4 enzymes, however, an interesting exception is LM2. The compounds bound stronger to pseudolysin than to thermolysin, and the molecular modelling studies showed that occupation of the S2(') subpocket by an aromatic group is favourable for strong interactions with pseudolysin.

Additivity or cooperativity: which model can predict the influence of simultaneous incorporation of two or more functionalities in a ligand molecule?

Predicting how binding affinity responds to ligand structural modifications in structure-activity relationship studies (SAR) is a major challenge in medicinal chemistry. This is particularly true when two or more of these modifications are carried out simultaneously. In this study, we present binding affinity data from several series of thermolysin inhibitors in which simultaneous structural modifications were investigated to determine whether they are cooperative or additive. Data revealed that, while additivity is at work in some cases, cooperativity is more commonly demonstrated. Cooperativity and additivity were then correlated with ligand descriptors, such as the spacing and the topological features of the modified groups, in a manner that may provide guidance as to when each model should be utilized. Cooperativity was particularly associated with contiguous groups and small unbranched hydrophobic side chain. Additivity, on the other hand, was associated with moderately distant hydrophobic group combinations and side chain branching. Such correlations can improve the predictability of SAR studies and can provide a starting point for additional investigations that may lead to further significant enhancements in the current scoring functions.

Inhibition of pseudolysin and thermolysin by hydroxamate-based MMP inhibitors.

In the present study, we have investigated the inhibition of thermolysin and pseudolysin by a series of compounds previously identified as matrix metalloproteinase (MMP) inhibitors using experimental binding studies and theoretical calculations. The experimental studies showed that some of the compounds were able to inhibit thermolysin and pseudolysin in the low µM range. The studies revealed that, in general, the compounds bound in the order MMPs > pseudolysin > thermolysin, and the strongest pseudolysin and thermolysin binders were compounds 8-12. Furthermore, compounds 8 and 9 were unique in that they bound much stronger to the two bacterial enzymes than to the MMPs. The docking calculations suggested that the phenyl group of the strongest binders (compounds 8 and 9) occupy the S2(')-subpocket, while a second ring system occupy the S1-subpocket in both thermolysin and pseudolysin. When the compounds possess two ring systems, the largest and most electron rich ring system seems to occupy the S1-subpocket.

Product inhibition in native-state proteolysis.

The proteolysis kinetics of intact proteins by nonspecific proteases provides valuable information on transient partial unfolding of proteins under native conditions. Native-state proteolysis is an approach to utilize the proteolysis kinetics to assess the energetics of partial unfolding in a quantitative manner. In native-state proteolysis, folded proteins are incubated with nonspecific proteases, and the rate of proteolysis is determined from the disappearance of the intact protein. We report here that proteolysis of intact proteins by nonspecific proteases, thermolysin and subtilisin deviates from first-order kinetics. First-order kinetics has been assumed for the analysis of native-state proteolysis. By analyzing the kinetics of proteolysis with varying concentrations of substrate proteins and also with cleavage products, we found that the deviation from first-order kinetics results from product inhibition. A kinetic model including competitive product inhibition agrees well with the proteolysis time course and allows us to determine the uninhibited rate constant for proteolysis as well as the apparent inhibition constant. Our finding suggests that the likelihood of product inhibition must be considered for quantitative assessment of proteolysis kinetics.

Methyl, ethyl, propyl, butyl: futile but not for water, as the correlation of structure and thermodynamic signature shows in a congeneric series of thermolysin inhibitors.

Water is ubiquitously present in any biological system and has therefore to be regarded as an additional binding partner in the protein-ligand binding process. Upon complex formation, a new solvent-exposed surface is generated and water molecules from the first solvation layer will arrange around this newly formed surface. So far, the influence of such water arrangements on the ligand binding properties is unknown. In this study, the binding modes of nine congeneric phosphonamidate-type inhibitors with systematically varied, size-increasing hydrophobic P2 ' substituents (from methyl to phenylethyl) addressing the hydrophobic, solvent-exposed S2 ' pocket of thermolysin were analyzed by high-resolution crystal structures and correlated with their thermodynamic binding profiles as measured by isothermal titration calorimetry. Overall, ΔΔG spreads over 7.0 kJ mol(-1) , ΔΔH varies by 15.8 kJ mol(-1) , and -TΔΔS by 12.1 kJ mol(-1) . Throughout the series, these changes correlate remarkably well with the geometric differences of water molecules arranged adjacent to the P2 ' substituents. Ligands with medium-sized P2 ' substituents exhibit highest affinities, presumably because of their optimal solvation patterns around these complexes. The addition, removal, or rearrangement of even a single methyl group can result in a strong modulation of the adjacent water network pattern shifting from enthalpy to entropy-driven binding. In conclusion, the quality of a water network assembled around a protein-ligand complex influences the enthalpy/entropy signature and can even modulate affinity to a surprising extent.

Synthesis and enzymatic evaluation of phosphoramidon and its ß anomer: Anomerization of α-l-rhamnose triacetate upon phosphitylation.

A novel and efficient strategy for the synthesis of phosphoramidon and its ß anomer has been developed by manipulating the anomerization of α-l-rhamnose triacetate upon phosphitylation. The experimental results suggest that proton transfer, bond rotation, and N atom are the key factors for the anomerization. The determined Ki and Kd values establish that phosphoramidon prepared by this method possesses excellent biological activity, and indicate that the contacts of rhamnose moiety with the enzyme have limited contribution to the binding.

Dipeptide inhibitors of thermolysin and angiotensin I-converting enzyme.

Thermolysin (TLN) and other thermolysin-like zinc metalloproteinases (TLPs),are important virulence factors for pathogenesis of bacterial infections by suppressing the innate immune system of the host. Therapeutic inhibition ofTLPs is believed to be a novel strategy inthe development of a new generation antibiotics.In the present study inhibition of TLN and angiotensin I-converting enzyme (ACE) by small peptides were studied by in vitro binding assays and theoretical calculations. The capacity of the peptides to inhibitTLN induced cleavage ofthe transcription factor nuclear factor kappa beta (NF-κB) was studied by electrophoretic mobility shift assays (EMSAs).Nine peptides inhibited ACE with IC50 values in the range 0.48 (IVY) to 1408 (HF) µM, while seven inhibited TLN with IC50 values in the range 0.00034 (IY) to 95640 (FW) µM. Calculations indicated that the peptides occupied the S1' and S2' subsites of ACE, and that IY, LW and IW occupiedthe S1' and S2' subsites, while FW, WL and WV occupiedthe S1 and S1' subsites of TLN. EMSA showed that peptides inhibited TLN induced cleavage of NF-κB. The studied peptides may form as a basis for the design of new compoundstargeting TLN with a potential in the treatment of bacterial infections.

Water mediated ligand functional group cooperativity: the contribution of a methyl group to binding affinity is enhanced by a COO(-) group through changes in the structure and thermodynamics of the hydration waters of ligand-thermolysin complexes.

Ligand functional groups can modulate the contributions of one another to the ligand-protein binding thermodynamics, producing either positive or negative cooperativity. Data presented for four thermolysin phosphonamidate inhibitors demonstrate that the differential binding free energy and enthalpy caused by replacement of a H with a Me group, which binds in the well-hydrated S2' pocket, are more favorable in presence of a ligand carboxylate. The differential entropy is however less favorable. Dissection of these differential thermodynamic parameters, X-ray crystallography, and density-functional theory calculations suggest that these cooperativities are caused by variations in the thermodynamics of the complex hydration shell changes accompanying the H→Me replacement. Specifically, the COO(-) reduces both the enthalpic penalty and the entropic advantage of displacing water molecules from the S2' pocket and causes a subsequent acquisition of a more enthalpically, less entropically, favorable water network. This study contributes to understanding the important role water plays in ligand-protein binding.

Water makes the difference: rearrangement of water solvation layer triggers non-additivity of functional group contributions in protein-ligand binding.

The binding of four congeneric peptide-like thermolysin inhibitors has been studied by high-resolution crystal structure analysis and isothermal titration calorimetry. The ligands differ only by a terminal carboxylate and/or methyl group. A surprising non-additivity of functional group contributions for the carboxylate and/or methyl groups is detected. Adding the methyl first and then the carboxylate group results in a small Gibbs free energy increase and minor enthalpy/entropy partitioning for the first modification, whereas the second involves a strong affinity increase combined with large enthalpy/entropy changes. However, first adding the carboxylate and then the methyl group yields reverse effects: the acidic group attachment now causes minor effects, whereas the added methyl group provokes large changes. As all crystal structures show virtually identical binding modes, affinity changes are related to rearrangements of the first solvation layer next to the S(2)' pocket. About 20-25 water molecules are visible next to the studied complexes. The added COO(-) groups perturb the local water network in both carboxylated complexes, and the attached methyl groups provide favorable interaction sites for water molecules. Apart from one example, a contiguously connected water network between protein and ligand functional groups is observed in all complexes. In the complex with the carboxylated ligand, which still lacks the terminal methyl group, the water network is unfavorably ruptured. This results in a surprising thermodynamic signature showing only a minor affinity increase upon COO(-) group attachment. Because the further added methyl group provides a favorable interaction site for water, the network can be reestablished, and a strong affinity increase with a large enthalpy/entropy signature is then detected.

Binding modes and pharmacophore modelling of thermolysin inhibitors.

In the present paper 25 known thermolysin inhibitors were docked into thermolysin using the Internal Coordinate Mechanics (ICM) software. Pharmacophore models based on thermolysin binding modes and activity profiles were generated using the LigandScout program. The docking studies indicated that all 25 inhibitors coordinated the catalytic zinc in bidentate or monodentate geometry. A 'three-point' pharmacophore model was proposed which consisted of a hydrophobic group, a negative ionizable group and a hydrogen bond acceptor group. Finally the pharmacophore model has been tested against a small compound library containing 18 highly, moderately, less active as well as inactive compounds. The screening indicated that the pharmacophore model could, identify highly active compounds in front of inactive or less active ones.

Specific interactions and binding energies between thermolysin and potent inhibitors: molecular simulations based on ab initio molecular orbital method.

Biochemical functions of the metalloprotease thermolysin (TLN) are controlled by various inhibitors. In a recent study we identified 12 compounds as TLN inhibitors by virtual screening and in vitro competitive binding assays. However, the specific interactions between TLN and these inhibitors have not been clarified. We here investigate stable structures of the solvated TLN-inhibitor complexes by classical molecular mechanics simulations and elucidate the specific interactions between TLN and these inhibitors at an electronic level by using ab initio fragment molecular orbital (FMO) calculations. The calculated binding energies between TLN and the inhibitors are qualitatively consistent with the experimental results, and the FMO results elucidate important amino acid residues of TLN for inhibitor binding. Based on the calculated results, we propose a novel potent inhibitor having a large binding affinity to TLN.

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions.

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.

Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases.

Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.

Identification of novel quinazolin-4(3H)-ones as inhibitors of thermolysin, the prototype of the M4 family of proteinases.

A combinatorial series of novel quinazolin-4(3H)-ones were synthesised and their structures were established based on spectroscopic data (IR, NMR, EI-MS, and FAB-MS). The compounds were tested for inhibition of the zinc metalloproteinase thermolysin (TLN) utilizing a chemical array-based approach. Some of the compounds were found to inhibit TLN, with IC(50) values ranging from 0.0115 microM (compound 3) to 122,637 microM (compound 29). Compound 3 [3-phenyl-2-(trifluoromethyl) quinazolin-4(3H)-one] (IC(50)=0.0115 microM) and compound 35 [3-(isopropylideneamino)-2,2-dimethyl-2,3-dihydroquinazolin-4 (1H)-one] (IC(50)=0.2477 microM) were found to be the most potent inhibitors.