Łukasz Berlicki

Remarkable potential of the α-aminophosphonate/phosphinate structural motif in medicinal chemistry.

R-Aminophosphonic acids are broadly defined as analogues of amino acids in which the carboxylic group is replaced by a phosphonic acid or related group (usually phosphonous or phosphinic acids). This results in the presence of the characteristic N C P scaffold (Scheme 1). The biological activity and natural occurrence of these compounds (often called R-aminophosphonates) were discovered half a century ago. Since then, the chemistry and biology of this class of compounds have been developed into a distinct branch of phosphorus chemistry. It is generally acknowledged that R-aminophosphonates possess a broad capability of influencing physiologic and pathologic processes, with applications ranging from agrochemistry to medicine. In some cases, these compounds have been commercialized. A number of excellent reviews on various aspects of their activity in natural systems have been published. 12 The mode of action of aminophosphonates primarily involves the inhibition of enzymes of different class and origin. Despite its long history, this area of research remains intensively explored and frequently delivers new promising lead compounds in medicinal chemistry. The N C P molecular fragment and its chemistry offer many possibilities for structural modifications, which have resulted in broad biological relevance (Scheme 1). Often, R-aminophosphonic and phosphinic acids are considered simple analogues of their natural counterparts, carboxylic acids. Although carboxylic and phosphonic acid groups differ in shape (tetrahedral at phosphorus versus planar at carbon), acidity (with phosphonic acid being significantly more acidic), and steric bulk (the phosphorus atom has a much larger atomic radius than carbon), they frequently exhibit similar properties, with the phosphonic acid being recognized by enzymes or receptors as false substrates or inhibitors. However, the tetrahedral geometry of substituents around the phosphorus moiety causes it to resemble the high-energy transition state (TS) of ester and amide bond hydrolyses. The tetrahedral transition state is believed to be specifically stabilized in enzyme active sites, which has inspired numerous studies on their applications in regulating the activity of proteases. This approach has been most successful in the case of metalloproteases, which have an organophosphorus moiety in their active sites that facilitates the chelation of metal ions. This approach has resulted in the development of many potent inhibitors of various enzymes, such as the antihypertensive drug fosinopril, an angiotensin I converting enzyme (ACE) inhibitor. Recently, the N C P scaffold has been used to construct extended transition state analogues of amide bond synthesis or hydrolysis to find potent inhibitors of enzymes such as glutamine synthetase or urease. Reactive peptidyl phosphonate diaryl esters have been successfully used to covalently modify members of the serine hydrolase superfamily. This approach exploits their ability to phosphonylate the hydroxyl residue of the active-site serine of these enzymes. They act as competitive, irreversible inhibitors, which, after the formation of an initial enzyme substrate complex, bind to the active site via a transesterification reaction and thus block its catalytic function. The activity and selectivity of the interactions of inhibitors with target enzymes can be adjusted by structural optimization of the S1 residues and/or by the development of an extended peptide chain. Finally, aminomethylenebisphosphonic acids form a separate class of medicinally important compounds bearing the N C P skeleton. They are hydrolytically stable analogues of pyrophosphate characterized by a common P C P fragment in which a carbon phosphorus bond replaces an oxygen phosphorus bond. Their primary medical application is in combating osteoporosis. They exhibit very high affinity to bone tissue, being rapidly adsorbed at the bone surface, and they regulate the bone remodeling process. Because the action of bisphosphonates is limited to osseous tissue, they have also been used to deliver conjugated chemotherapeutic agents to bone. Likely because of their strong chelating properties, bisphosphonates also exhibit inhibitory properties toward a wide variety of metalloenzymes. In this Perspective, we present the key features of theN C P molecular fragment that govern the activity of the molecules that incorporate it. A general overview of known modes of action and target enzyme classes is briefly presented. Recent representative medicinal chemistry projects are described and discussed, including the achievements of our research group on leucine aminopeptidase and urease. Particular attention is given to the molecular aspects of the N C P mechanism of action and to the rational design of new compounds based on threedimensional structures. The potential future applications of this class of compounds are also discussed.

Urease inhibitors as potential drugs for gastric and urinary tract infections: a patent review

Introduction: Urease is the enzyme that catalyzes the hydrolysis of urea, which is involved in serious infections caused by Helicobacter pylori in the gastric tract, as well as Proteus and related species in the urinary tract. The necessity to treat such infections has stimulated intensive studies on various groups of urease inhibitors. Areas covered: Patent literature on urease inhibitors with possible applications in medicine is reviewed in this paper. Hydroxamic acids, phosphoramidates, urea derivatives, quinones and heterocyclic compounds constitute the major classes of structures with such activity. Expert opinion: Until now, only one compound, acetohydroxamic acid, has been clinically used for the treatment of urinary tract infections by urease inhibition. Unfortunately, it exhibits severe side effects. Thus, it seems that the full potential of urease inhibition has not yet been fully explored. Several Japanese patents related to the use of herbal extracts as sources of polyphenolic urease inhibitors have been considered as complementary or alternative therapy; however, their accessibility is quite possibly due to reduced restrictions for the introduction of natural products to the market.

Peptides containing β-amino acid patterns: challenges and successes in medicinal chemistry.

The construction of bioactive peptides using β-amino acid-containing sequence patterns is a very promising strategy to obtain analogues that exhibit properties of high interest for medicinal chemistry applications. β-Amino acids have been shown to modulate the conformation, dynamics, and proteolytic susceptibility of native peptides. They can be either combined with α-amino acids by following specific patterns, which results in backbone architectures with well-defined orientations of the side chain functional groups, or assembled in de novo-designed bioactive β- or α,β-peptidic sequences. Such peptides display various biological functions, including antimicrobial activity, inhibition of protein-protein interactions, agonism/antagonism of GPCR ligands, and anti-angiogenic activity.

Peptide-based inhibitors of protein-protein interactions.

Protein-protein interactions (PPIs) are key elements of several important biological processes and have emerged as valuable targets in medicinal chemistry. Importantly, numerous specific protein-protein interactions (e.g., p53-HDM2 and Bcl-2-BH3 domains) were found to be involved in the development of several diseases, including various types of cancer. In general, the discovery of new synthetic PPI inhibitors is a challenging task because protein surfaces have not evolved in a manner that allows for specific binding of low molecular weight compounds. Here, we review the discovery strategies for peptide-based PPI inhibitors. Although peptide-based drug candidates exhibit significant drawbacks (in particular, low proteolytic stability), modifications of either the side chains or backbone could provide molecules of interest. Moreover, due to the large molecular size of peptide-based compounds, the discovery of molecules that specifically interact with extended protein surfaces is possible. Two major strategies for constructing peptide-based PPI inhibitors are as follows: (a) cyclization (e.g., stapled peptides) and (b) modification of the backbone structure (e.g., β-peptides and peptoids). These approaches for constructing PPI inhibitors enhance both the inhibitory activity and pharmacokinetic properties compared with non-modified α-peptides.

Effectiveness and mode of action of phosphonate inhibitors of plant glutamine synthetase.

BACKGROUND Aiming at the rational design of new herbicides, the availability of the three-dimensional structure of the target enzyme greatly enhances the optimisation of lead compounds and the design of derivatives with increased activity. Among the most widely exploited herbicide targets is glutamine synthetase. Recently, the structure of a cytosolic form of the maize enzyme has been described, making it possible to verify whether steric, electronic and hydrophobic features of a compound are in agreement with inhibitor-protein interaction geometry. RESULTS Three series of compounds (aminophosphonates, hydroxyphosphonates and aminomethylenebisphosphonates) were evaluated as possible inhibitors of maize glutamine synthetase. Aminomethylenebisphosphonate derivatives substituted in the phenyl ring retained the inhibitory potential, whereas variations in the scaffold, i.e. the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of activity. A kinetic characterisation showed a non-competitive mechanism against glutamate and an uncompetitive mechanism against ATP. A docking analysis suggested the mode of bisphosphonate binding to the active site. CONCLUSION Results made it possible to define the features required to maintain or enhance the biological activity of these compounds, which represent lead structures to be further exploited for the design of new substances endowed with herbicidal activity.

Enantiodifferentiation of aminophosphonic and aminophosphinic acids with α- and β-cyclodextrins

Abstract Cyclodextrins were used as chiral selectors for the 31 P NMR determination of the enantiomeric excess of aminoalkanephosphonic and aminoalkanephosphinic acids. Most of these acids form inclusion complexes with α- and/or β-cyclodextrin and upon increasing the cyclodextrin to aminophosphonic acid molar ratio 31 P NMR signals for ( R )- and ( S )-enantiomers separate. ROESY spectra allowed the determination of structures of the inclusion complexes.

The crystal structure of Sporosarcina pasteurii urease in a complex with citrate provides new hints for inhibitor design

Urease, the enzyme that catalyses the hydrolysis of urea, is a virulence factor for a large number of ureolytic bacterial human pathogens. The increasing resistance of these pathogens to common antibiotics as well as the need to control urease activity to improve the yield of soil nitrogen fertilization in agricultural applications has stimulated the development of novel classes of molecules that target urease as enzyme inhibitors. We report on the crystal structure at 1.50-Å resolution of a complex formed between citrate and urease from Sporosarcina pasteurii, a widespread and highly ureolytic soil bacterium. The fit of the ligand to the active site involves stabilizing interactions, such as a carboxylate group that binds the nickel ions at the active site and several hydrogen bonds with the surrounding residues. The citrate ligand has a significantly extended structure compared with previously reported ligands co-crystallized with urease and thus represents a unique and promising scaffold for the design of new, highly active, stable, selective inhibitors.

Replacement of Thr32 and Gln34 in the C-terminal neuropeptide Y fragment 25-36 by cis-cyclobutane- and cis-cyclopentane β-amino acids shifts selectivity toward the Y4 receptor

Neuropeptide Y (NPY) and pancreatic polypeptide (PP) control central and peripheral processes by activating the G protein coupled receptors YxR (x = 1, 2, 4, 5). We present analogs of the C-terminal fragments 25-36 and 32-36 of NPY and PP containing (1R,2S)-cyclobutane (βCbu) or (1R,2S)-cyclopentane (βCpe) β-amino acids, which display exclusively Y4R affinity. In particular, [βCpe(34)]-NPY-(25-36) is a Y4R selective partial agonist (EC50 41 ± 6 nM, Emax 71%) that binds Y4R with a Ki of 10 ± 2 nM and a selectivity >100-fold relative to Y1R and Y2R and >50-fold relative to Y5R. Comparably, [Y(32), βCpe(34)]-NPY(PP)-(32-36) selectively binds and activates Y4R (EC50 94 ± 21 nM, Emax 73%). The NMR structure of [βCpe(34)]-NPY-(25-36) in dodecylphosphatidylcholine micelles shows a short helix at residues 27-32, while the C-terminal segment R(33)βCpe(34)R(35)Y(36) is extended. The biological properties of the βCbu- or βCpe-containing NPY and PP C-terminal fragments encourage the future application of these β-amino acids in the synthesis of selective Y4R ligands.

1,2-Benzisoselenazol-3(2H)-one Derivatives As a New Class of Bacterial Urease Inhibitors

Urease inhibitors are considered promising compounds for the treatment of ureolytic bacterial infections, particularly infections resulting from Helicobacter pylori in the gastric tract. Herein, we present the synthesis and the inhibitory activity of novel and highly effective organoselenium compounds as inhibitors of Sporosarcina pasteurii and Helicobacter pylori ureases. These studied compounds represent a class of competitive reversible urease inhibitors. The most active compound, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen), displayed Ki values equal to 2.11 and 226 nM against S. pasteurii and H. pylori enzymes, respectively, indicating ebselen as one of the most potent low-molecular-weight inhibitors of bacterial ureases reported to date. Most of these molecules penetrated through the cell membrane of the Gram-negative bacteria Escherichia coli (pGEM::ureOP) in vitro. Furthermore, whole-cell studies on the H. pylori J99 reference strain confirmed the high efficiency of the examined organoselenium compounds as urease inhibitors against pathogenic bacteria.

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.