Artur Mucha

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.

Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development.

Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs.

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH2]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.

Metallo-aminopeptidase inhibitors.

Abstract Aminopeptidases are enzymes that selectively hydrolyze an amino acid residue from the N-terminus of proteins and peptides. They are important for the proper functioning of prokaryotic and eukaryotic cells, but very often are central players in the devastating human diseases like cancer, malaria and diabetes. The largest aminopeptidase group include enzymes containing metal ion(s) in their active centers, which often determines the type of inhibitors that are the most suitable for them. Effective ligands mostly bind in a non-covalent mode by forming complexes with the metal ion(s). Here, we present several approaches for the design of inhibitors for metallo-aminopeptidases. The optimized structures should be considered as potential leads in the drug discovery process against endogenous and infectious diseases.

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.

The m18 aspartyl aminopeptidase of the human malaria parasite Plasmodium falciparum

A member of the M18 family of aspartyl aminopeptidases is expressed by all intra-erythrocytic stages of the human malaria parasite Plasmodium falciparum (PfM18AAP), with highest expression levels in rings. Functionally active recombinant enzyme, rPfM18AAP, and native enzyme in cytosolic extracts of malaria parasites are 560-kDa octomers that exhibit optimal activity at neutral pH and require the presence of metal ions to maintain enzymatic activity and stability. Like the human aspartyl aminopeptidase, the exopeptidase activity of PfM18AAP is exclusive to N-terminal acidic amino acids, glutamate and aspartate, making this enzyme of particular interest and suggesting that it may function alongside the malaria cytosolic neutral aminopeptidases in the release of amino acids from host hemoglobin-derived peptides. Whereas immunocytochemical studies using transgenic P. falciparum parasites show that PfM18AAP is expressed in the cytosol, immunoblotting experiments revealed that the enzyme is also trafficked out of the parasite into the surrounding parasitophorous vacuole. Antisense-mediated knockdown of PfM18AAP results in a lethal phenotype as a result of significant intracellular damage and validates this enzyme as a target at which novel antimalarial drugs could be directed. Novel phosphinic derivatives of aspartate and glutamate showed modest inhibition of rPfM18AAP but did not inhibit malaria growth in culture. However, we were able to draw valuable observations concerning the structure-activity relationship of these inhibitors that can be employed in future inhibitor optimization studies.

High-performance liquid chromatographic enantiomer separation and determination of absolute configurations of phosphinic acid analogues of dipeptides and their α-aminophosphinic acid precursors

The enantiomers of N-benzyloxycarbonyl-phosphinic pseudodipeptides and their N-benzyloxycarbonyl-α-aminophosphinic acid precursors as well as various other structural analogues were separated on a set of cinchona alkaloid-derived chiral anion-exchangers by HPLC in the reversed-phase mode. Semi-preparative scale chromatography provided single enantiomers in 100 mg quantities. The configurations of the enantiomers were assigned indirectly by enantioselective chromatography on the basis of the elution order and was confirmed by enantiomeric reference compounds.

The preparation of phosphono peptides containing a phosphonamidate bond

Abstract The synthesis of phosphono peptides containing phosphonamidate bond by means of phosphorylation of amino acid esters with N-protected aminoalkylphosphonochloridates, is accompanied by the formation of an unidentified side-product. The factors influencing this reaction were studied in some detail.

Synthesis and Modifications of Phosphinic Dipeptide Analogues

Pseudopeptides containing the phosphinate moiety (-P(O)(OH)CH2-) have been studied extensively, mainly as transition state analogue inhibitors of metalloproteases. The key synthetic aspect of their chemistry is construction of phosphinic dipeptide derivatives bearing appropriate side-chain substituents. Typically, this synthesis involves a multistep preparation of two individual building blocks, which are combined in the final step. As this methodology does not allow simple variation of the side-chain structure, many efforts have been dedicated to the development of alternative approaches. Recent achievements in this field are summarized in this review. Improved methods for the formation of the phosphinic peptide backbone, including stereoselective and multicomponent reactions, are presented. Parallel modifications leading to the structurally diversified substituents are also described. Finally, selected examples of the biomedical applications of the title compounds are given.

Individual stereoisomers of phosphinic dipeptide inhibitor of leucine aminopeptidase.

Individual stereoisomers of the phosphinic pseudodipeptide hPhepsi[P(O)(OH)CH(2)]Phe were obtained by stereoselective liquid chromatographic separation as N- and C-terminally protected derivative on quinidine carbamate modified silica stationary phase. The stereoisomeric purity, exceeding 95% for each fraction, was determined before and after deprotection using two independent methods. The absolute configuration was rationally assigned by application of enantiomerically pure phosphinic acid substrates in the synthetic procedure and correlation with biological activity of the products. Substantial differences in inhibition of leucine aminopeptidase by the individual isomers revealed novel insights into potency of the recently developed and remarkably effective compound.