Inhibitory activity of catecholic phosphonic and phosphinic acids against Helicobacter pylori ureolysis.

Catechols have been reported to be potent covalent inhibitors of ureases, and they exhibit activity by modifying cysteine residues at the entrance to enzymatic active sites. Following these principles, we designed and synthesized novel catecholic derivatives that contained carboxylate and phosphonic/phosphinic functionalities and assumed expanded specific interactions. When studying the chemical stability of the molecules, we found that their intrinsic acidity catalyzes spontaneous esterification/hydrolysis reactions in methanol or water solutions, respectively. Regarding biological activity, the most promising compound, 2-(3,4-dihydroxyphenyl)-3-phosphonopropionic acid (15), exhibited significant anti-urease potential (Ki = 2.36 µM, Sporosarcinia pasteurii urease), which was reflected in the antiureolytic effect in live Helicobacter pylori cells at a submicromolar concentration (IC50 = 0.75 µM). As illustrated by molecular modeling, this compound was bound in the active site of urease through a set of concerted electrostatic and hydrogen bond interactions. The antiureolytic activity of catecholic phosphonic acids could be specific because these compounds were chemically inert and not cytotoxic to eukaryotic cells.

Optimized Ebselen-Based Inhibitors of Bacterial Ureases with Nontypical Mode of Action.

Screening of 25 analogs of Ebselen, diversified at the N-aromatic residue, led to the identification of the most potent inhibitors of Sporosarcina pasteurii urease reported to date. The presence of a dihalogenated phenyl ring caused exceptional activity of these 1,2-benzisoselenazol-3(2H)-ones, with Ki value in a low picomolar range (<20 pM). The affinity was attributed to the increased π-π and π-cation interactions of the dihalogenated phenyl ring with αHis323 and αArg339 during the initial step of binding. Complementary biological studies with selected compounds on the inhibition of ureolysis in whole Proteus mirabilis cells showed a very good potency (IC50 < 25 nM in phosphate-buffered saline (PBS) buffer and IC90 < 50 nM in a urine model) for monosubstituted N-phenyl derivatives. The crystal structure of S. pasteurii urease inhibited by one of the most active analogs revealed the recurrent selenation of the Cys322 thiolate, yielding an unprecedented Cys322-S-Se-Se chemical moiety.

Antibacterial Activity of Ebselen.

Ebselen is a low-molecular-weight organoselenium compound that has been broadly studied for its antioxidant, anti-inflammatory, and cytoprotective properties. These advantageous properties were initially associated with mimicking the activity of selenoprotein glutathione peroxidase, but the biomedical impact of this compound appear to be far more complex. Ebselen serves as a substrate or inhibitor with multiple protein/enzyme targets, whereas inhibition typically originates from the covalent modification of cysteine residues by opening the benzisoselenazolone ring and S-Se bond formation. The inhibition of enzymes of various classes and origins has been associated with substantial antimicrobial potential among other activities. In this contribution, we summarize the current state of the art regarding the antibacterial activity of ebselen. This activity, alone and in combination with commercial pharmaceuticals, against pathogens, including those resistant to drugs, is presented, together with the molecular mechanism behind the reactivity. The specific inactivation of thioredoxin reductase, bacterial toxins, and other resistance factors is considered to have certain therapeutic implications. Synergistic action and sensitization to common antibiotics assisted with the use of ebselen appear to be promising directions in the treatment of persistent infections.

Covalent Inhibition of Bacterial Urease by Bifunctional Catechol-Based Phosphonates and Phosphinates.

In this study, a new class of bifunctional inhibitors of bacterial ureases, important molecular targets for antimicrobial therapies, was developed. The structures of the inhibitors consist of a combination of a phosphonate or (2-carboxyethyl)phosphinate functionality with a catechol-based fragment, which are designed for complexation of the catalytic nickel ions and covalent bonding with the thiol group of Cys322, respectively. Compounds with three types of frameworks, including ß-3,4-dihydroxyphenyl-, α-3,4-dihydroxybenzyl-, and α-3,4-dihydroxybenzylidene-substituted derivatives, exhibited complex and varying structure-dependent kinetics of inhibition. Among irreversible binders, methyl ß-(3,4-dihydroxyphenyl)-ß-(2-carboxyethyl)phosphorylpropionate was observed to be a remarkably reactive inhibitor of Sporosarcina pasteurii urease (kinact/KI = 10 420 s-1 M-1). The high potential of this group of compounds was also confirmed in Proteus mirabilis whole-cell-based inhibition assays. Some compounds followed slow-binding and reversible kinetics, e.g., methyl ß-(3,4-dihydroxyphenyl)-ß-phosphonopropionate, with Ki* = 0.13 µM, and an atypical low dissociation rate (residence time τ = 205 min).

N-Benzyl Residues as the P1' Substituents in Phosphorus-Containing Extended Transition State Analog Inhibitors of Metalloaminopeptidases.

Peptidyl enzyme inhibitors containing an internal aminomethylphosphinic bond system (P(O)(OH)-CH2-NH) can be termed extended transition state analogs by similarity to the corresponding phosphonamidates (P(O)(OH)-NH). Phosphonamidate pseudopeptides are broadly recognized as competitive mechanism-based inhibitors of metalloenzymes, mainly hydrolases. Their practical use is, however, limited by hydrolytic instability, which is particularly restricting for dipeptide analogs. Extension of phosphonamidates by addition of the methylene group produces a P-C-N system fully resistant in water conditions. In the current work, we present a versatile synthetic approach to such modified dipeptides, based on the three-component phospha-Mannich condensation of phosphinic acids, formaldehyde, and N-benzylglycines. The last-mentioned component allowed for simple and versatile introduction of functionalized P1' residues located on the tertiary amino group. The products demonstrated moderate inhibitory activity towards porcine and plant metalloaminopeptidases, while selected derivatives appeared very potent with human alanyl aminopeptidase (Ki = 102 nM for 6a). Analysis of ligand-protein complexes obtained by molecular modelling revealed canonical modes of interactions for mono-metallic alanyl aminopeptidases, and distorted modes for di-metallic leucine aminopeptidases (with C-terminal carboxylate, not phosphinate, involved in metal coordination). In general, the method can be dedicated to examine P1'-S1' complementarity in searching for non-evident structures of specific residues as the key fragments of perspective ligands.

P1' Residue-Oriented Virtual Screening for Potent and Selective Phosphinic (Dehydro) Dipeptide Inhibitors of Metallo-Aminopeptidases.

Designing side chain substituents complementary to enzyme binding pockets is of great importance in the construction of potent and selective phosphinic dipeptide inhibitors of metallo-aminopeptidases. Proper structure selection makes inhibitor construction more economic, as the development process typically consists of multiple iterative preparation/bioassay steps. On the basis of these principles, using noncomplex computation and modeling methodologies, we comprehensively screened 900 commercial precursors of the P1' residues of phosphinic dipeptide and dehydrodipeptide analogs to identify the most promising ligands of 52 metallo-dependent aminopeptidases with known crystal structures. The results revealed several nonproteinogenic residues with an improved energy of binding compared with the best known inhibitors. The data are discussed taking into account the selectivity and stereochemical implications of the enzymes. Using this approach, we were able to identify nontrivial structural elements substituting the recognized phosphinic peptidomimetic scaffold of metallo-aminopeptidase inhibitors.

Peptide and Pseudopeptide Bond Synthesis in Phosphorus Dipeptide Analogs.

Peptide analogs modified with a phosphorus-based moiety (phosphonate, phosphonamidate, or phosphinate) have emerged as invaluable tools in fundamental and medicinal, mechanistic, and inhibitory studies of proteolytic enzymes and other catalytic proteins that process the amino acids and peptides. The first stages of the chemical synthesis of these compounds frequently involve formation of peptide or pseudopeptide bond between a suitably protected α-amino acid and an α-aminoalkyl phosphorus derivative. These preparative protocols are distinct from conventional solution and solid-phase peptide syntheses that have become routine and automatized. In the following chapter, we describe in details the methods and techniques utilized to perform this nonstandard coupling and to obtain P-terminal dipeptidyl phosphonates and pseudodipeptides containing the internal phosphonamidate or phosphinate linkages. Methods of products' purification, the deprotection conditions, and stability issues are also presented and discussed.

Recent Developments in Peptidyl Diaryl Phoshonates as Inhibitors and Activity-Based Probes for Serine Proteases.

This review presents current achievements in peptidyl diaryl phosphonates as covalent, specific mechanism-based inhibitors of serine proteases. Along three decades diaryl phosphonates have emerged as invaluable tools in fundamental and applicative studies involving these hydrolases. Such an impact has been promoted by advantageous features that characterize the phosphonate compounds and their use. First, the synthesis is versatile and allows comprehensive structural modification and diversification. Accordingly, reactivity and specificity of these bioactive molecules can be easily controlled by appropriate adjustments of the side chains and the leaving groups. Secondly, the phosphonates target exclusively serine proteases and leave other oxygen and sulfur nucleophiles intact. Synthetic accessibility, lack of toxicity, and promising pharmacokinetic properties make them good drug candidates. In consequence, the utility of peptidyl diaryl phosphonates continuously increases and involves novel enzymatic targets and innovative aspects of application. For example, conjugation of the structures of specific inhibitors with reporter groups has become a convenient approach to construct activity-based molecular probes capable of monitoring location and distribution of serine proteases.

Recent developments in the synthesis and applications of phosphinic peptide analogs.

Synthetic pseudopeptides that fit well with the active site architecture allow the most effective binding to enzymes, similar to native substrates in high-energy transition states. Phosphinic acid peptide analogs that comprise the tetrahedral phosphorus moiety introduced to replace an internal amide bond exert such an isosteric or isoelectronic resemblance, combined with providing other advantageous features, for example, metal complexing properties. Accordingly, they are capable of inhibiting metal-dependent enzymes involved in biological functions in eukaryotic and prokaryotic cells. These enzymes are associated with notorious human diseases, such as cancer, e.g., matrix metalloproteinases, or are etiological factors of protozoal and bacterial infections, e.g., metalloaminopeptidases. The affinity and selectivity of these compounds can be conveniently adjusted, either by structural modification of dedicated side chains or by backbone elongation to enhance specific interactions with the corresponding binding pockets. Recent approaches to the synthesis of these compounds are illustrated by examples of the preparation of rationally designed structures of inhibitors of particular enzymes. Activity against appealing enzymatic targets is presented, along with the molecular mechanisms of action and therapeutic implications. Innovative aspects of phosphinic peptide application, e.g., as activity-based probes, and ligands of complexes of radioisotopes for nuclear medicine are also outlined.

Bisphosphonic acids and related compounds as inhibitors of nucleotide- and polyphosphate-processing enzymes: A PPK1 and PPK2 case study.

Bisphosphonic acids, which are structural analogs of pyrophosphate, constitute a class of compounds with very high potential for the construction of effective inhibitors of enzymes operating on oligo- and polyphosphates. The bisphosphonate-based methodology was applied for the discovery of inhibitors of two families of polyphosphate kinases (PPK1 and PPK2). Screening of thirty-two structurally diverse bisphosphonic acids and related compounds revealed several micromolar inhibitors of both enzymes. Importantly, selectivity of bisphosphonates could be achieved by application of the appropriate side chain.

Structural exploration of cinnamate-based phosphonic acids as inhibitors of bacterial ureases.

The conjugated system of cinnamic acid, α-substituted with a phosphonoalkyl residue, was previously validated as a scaffold that provided one of the most potent organophosphorus inhibitors of bacterial urease. Following the idea of using Morita-Baylis-Hillman adducts to introduce the terminal phosphonic side chain functionality to the α,ß-unsaturated system, we currently report the synthesis and activity of an extended series of compounds. Cinnamates modified with 3-phosphonopropyl and 4-phosphonobutyl side chains were obtained in a convenient two-step procedure, which involved Pd-mediated transformations of the Morita-Baylis-Hillman bromides as the key substrates. The introduction of a terminal alkenyl fragment, which was achieved by Stille coupling with stannanes, was followed by a tandem C-P bond formation/oxidation process. A submicromolar ligand of Sporosarcina pasteurii urease (Ki = 0.509 µM) was identified among the active molecules. In addition, inhibitors of Proteus mirabilis urease affected bacterial growth at the micromolar level. Based on the structure-activity relationship and the mechanism of inhibition, we suggest a nontypical mixed mode of action for the slow binding compounds. We presume that the molecular distance between the phosphonic group and the backbone double bond allows a dual activity: complexation of the acidic group with nickel ions and Michael addition of a cysteine forming the active site lid.

Neutral metalloaminopeptidases APN and MetAP2 as newly discovered anticancer molecular targets of actinomycin D and its simple analogs.

The potent transcription inhibitor Actinomycin D is used with several cancers. Here, we report the discovery that this naturally occurring antibiotic inhibits two human neutral aminopeptidases, the cell-surface alanine aminopeptidase and intracellular methionine aminopeptidase type 2. These metallo-containing exopeptidases participate in tumor cell expansion and motility and are targets for anticancer therapies. We show that the peptide portions of Actinomycin D and Actinomycin X2 are not required for effective inhibition, but the loss of these regions changes the mechanism of interaction. Two structurally less complex Actinomycin D analogs containing the phenoxazone chromophores, Questiomycin A and Actinocin, appear to be competitive inhibitors of both aminopeptidases, with potencies similar to the non-competitive macrocyclic parent compound (Ki in the micromolar range). The mode of action for all four compounds and both enzymes was demonstrated by molecular modeling and docking in the corresponding active sites. This knowledge gives new perspectives to Actinomycin D's action on tumors and suggests new avenues and molecules for medical applications.

Aminophosphinates against Helicobacter pylori ureolysis-Biochemical and whole-cell inhibition characteristics.

Urease is an important virulence factor from Helicobacter pylori that enables bacterial colonization of human gastric mucosa. Specific inhibition of urease activity can be regarded as a promising adjuvant strategy for eradication of this pathogen. A group of organophosphorus inhibitors of urease, namely, aminophosphinic acid and aminophosphonic acid derivatives, were evaluated in vitro against H. pylori urease. The kinetic characteristics of recombinant enzyme activity demonstrated a competitive reversible mode of inhibition with Ki values ranging from 0.294 to 878 µM. N-n-Hexylaminomethyl-P-aminomethylphosphinic acid and N-methylaminomethyl-P-hydroxymethylphosphinic acid were the most effective inhibitors (Ki = 0.294 µM and 1.032 µM, respectively, compared to Ki = 23 µM for the established urease inhibitor acetohydroxamic acid). The biological relevance of the inhibitors was verified in vitro against a ureolytically active Escherichia coli Rosetta host that expressed H. pylori urease and against a reference strain, H. pylori J99 (CagA+/VacA+). The majority of the studied compounds exhibited urease-inhibiting activity in these whole-cell systems. Bis(N-methylaminomethyl)phosphinic acid was found to be the most effective inhibitor in the susceptibility profile studies of H. pylori J99. The cytotoxicity of nine structurally varied inhibitors was evaluated against four normal human cell lines and was found to be negligible.

Novel organophosphorus scaffolds of urease inhibitors obtained by substitution of Morita-Baylis-Hillman adducts with phosphorus nucleophiles.

The reactivity of Morita-Baylis-Hillman allyl acetates was employed to introduce phosphorus-containing functionalities to the side chain of the cinnamic acid conjugated system by nucleophilic displacement. The proximity of two acidic groups, the carboxylate and phosphonate/phosphinate groups, was necessary to form interactions in the active site of urease by recently described inhibitor frameworks. Several organophosphorus scaffolds were obtained and screened for inhibition of the bacterial urease, an enzyme that is essential for survival of urinary and gastrointestinal tract pathogens. α-Substituted phosphonomethyl- and 2-phosphonoethyl-cinnamate appeared to be the most potent and were further optimized. As a result, one of the most potent organophosphorus inhibitors of urease, α-phosphonomethyl-p-methylcinnamic acid, was identified, with Ki = 0.6 µM for Sporosarcina pasteurii urease. High complementarity to the enzyme active site was achieved with this structure, as any further modifications significantly decreased its affinity. Finally, this work describes the challenges faced in developing ligands for urease.

Identification of methionine aminopeptidase 2 as a molecular target of the organoselenium drug ebselen and its derivatives/analogues: Synthesis, inhibitory activity and molecular modeling study.

A collection of twenty-six organoselenium compounds, ebselen and its structural analogues, provided a novel approach for inhibiting the activity of human methionine aminopeptidase 2 (MetAP2). This metalloprotease, being responsible for the removal of the amino-terminal methionine from newly synthesized proteins, plays a key role in angiogenesis, which is essential for the progression of diseases, including solid tumor cancers. In this work, we discovered that ebselen, a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity, inhibits one of the main enzymes in the tumor progression pathway. Using three-step synthesis, we obtained twenty-five ebselen derivatives/analogues, ten of which are new, and tested their inhibitory activity toward three neutral aminopeptidases (MetAP2, alanine and leucine aminopeptidases). All of the tested compounds proved to be selective, slow-binding inhibitors of MetAP2. Similarly to ebselen, most of its analogues exhibited a moderate potency (IC50=1-12µM). Moreover, we identified three strong inhibitors that bind favorably to the enzyme with the half maximal inhibitory concentration in the submicromolar range.

Whole-cell Proteus mirabilis urease inhibition by aminophosphinates for the control of struvite formation.

The study evaluated the in vitro impact of a series of aminophosphinic urease inhibitors on Proteusmirabilis. The group of compounds comprised structurally diverse analogues of diamidophosphate built on an N-C-P scaffold. The influence of urease inhibition on urea-splitting activity was assessed by whole-cell pH-static kinetic measurements. The potential to prevent struvite formation was determined by monitoring changes in pH and ionic composition of artificial urine medium during P. mirabilis growth. The most active compounds exhibited stronger positive effect on urine stability than the acknowledged inhibitor acetohydroxamic acid. The high anti-ureolytic and pH-stabilizing effect of urease inhibitors 4 and 14 was well correlated with their reported kinetic properties against pure urease from P. mirabilis (Ki values of 0.62±0.09 and 0.202±0.057 µM, respectively, compared to 5.7±0.4 µM for acetohydroxamic acid). The effect of repressed ureolysis upon the viability of Proteus cells was studied using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] metabolic efficiency assay and LIVE/DEAD fluorescent staining. Most of the compounds caused whole-cell dehydrogenase activity loss; four structures (1, 2, 4 and 14) reduced the culture viability by nearly 70 % at 1 mM concentration. Results of dual fluorescent staining suggested that besides urea-splitting prevention, the structures additionally exerted an outer-membrane-destabilizing effect.

Discovery of potent and selective inhibitors of human aminopeptidases ERAP1 and ERAP2 by screening libraries of phosphorus-containing amino acid and dipeptide analogues.

A collection of fifty phosphonic and phosphinic acids was screened for inhibition of ERAP1 and ERAP2, the human endoplasmic reticulum aminopeptidases. The cooperative action of these enzymes is manifested by trimming a variety of antigenic precursors to be presented on the cell surface by major histocompatibility class I. The SAR studies revealed several potent compounds, particularly among the phosphinic dipeptide analogues, that were strong inhibitors of ERAP2 (Ki=100-350nM). A wide structural diversity of the applied organophosphorus compounds, predominantly non-proteinogenic analogues, allowed identification of representatives selective toward only one form of ERAP. For example, N'-substituted α,ß-diaminophosphonates and phosphinates exhibited potency only toward ERAP2, which is in agreement with the P1 basic substrate-oriented specificity. Such discriminating ligands are invaluable tools for elucidating the precise role of a particular aminopeptidase in the concerted function of antigen processing and in human diseases.

A structural insight into the P1S1 binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases.

N'-substituted 1,2-diaminoethylphosphonic acids and 1,2-diaminoethylphosphinic dipeptides were explored to unveil the structural context of the unexpected selectivity of these inhibitors of M1 alanine aminopeptidases (APNs) versus M17 leucine aminopeptidase (LAP). The diaminophosphonic acids were obtained via aziridines in an improved synthetic procedure that was further expanded for the phosphinic pseudodipeptide system. The inhibitory activity, measured for three M1 and one M17 metalloaminopeptidases of different sources (bacterial, human and porcine), revealed several potent compounds (e.g., Ki = 65 nM of 1u for HsAPN). Two structures of an M1 representative (APN from Neisseria meningitidis) in complex with N-benzyl-1,2-diaminoethylphosphonic acid and N-cyclohexyl-1,2-diaminoethylphosphonic acid were determined by the X-ray crystallography. The analysis of these structures and the models of the phosphonic acid complexes of the human ortholog provided an insight into the role of the additional amino group and the hydrophobic substituents of the ligands within the S1 active site region.

Zwitterionic phosphorylated quinines as chiral solvating agents for NMR spectroscopy.

Because of their unique 3D arrangement, naturally occurring Cinchona alkaloids and their synthetic derivatives have found wide-ranging applications in chiral recognition. Recently, we determined the enantioselective properties of C-9-phosphate mixed triesters of quinine as versatile chiral solvating agents in nuclear magnetic resonance (NMR) spectroscopy. In the current study, we introduce new zwitterionic members of this class of molecules containing a negatively charged phosphate moiety (i.e., ethyl, n-butyl and phenyl hydrogen quininyl phosphate). An efficient approach for synthesizing these compounds is elaborated, and full characterization, including conformational and autoaggregation phenomena studies, was performed. Therefore, their ability to induce NMR anisochrony of selected enantiomeric substrates (i.e., primarily N-DNB-protected amino acids and their methyl esters) was analyzed compared to uncharged diphenyl quininyl phosphate and its positively charged quaternary ammonium hydrochloride salt. In addition, (1) H and (13) C NMR experiments revealed their enantiodiscrimination potential toward novel analytes, such as secondary amines and nonprotected amino acids.

Bis(aminomethyl)phosphinic Acid, a Highly Promising Scaffold for the Development of Bacterial Urease Inhibitors.

Inhibitors of bacterial ureases are considered to be promising compounds in the treatment of infections caused by Helicobacter pylori in the gastric tract and/or by urealytic bacteria (e.g., Proteus species) in the urinary tract. A new, extended transition state scaffold, bis(aminomethyl)phosphinic acid, was successfully explored for the construction of effective enzyme inhibitors. A reliable methodology for the synthesis of phosphinate analogues in a three-component Mannich-type reaction was elaborated. The obtained molecules were assayed against ureases purified from Sporosarcina pasteurii and Proteus mirabilis, and aminomethyl(N-n-hexylaminomethyl)phosphinic acid was found to be the most potent inhibitor, with a K i = 108 nM against the S. pasteurii enzyme.