Compounds with a phosphonate group, i.e., -P(O)(OH)2 group attached directly to the molecule via a P-C bond serve as suitable non-hydrolyzable phosphate mimics in various biomedical applications. In principle, they often inhibit enzymes utilizing various phosphates as substrates. In this review we focus mainly on biologically active phosphonates that originated from our institute (Institute of Organic Chemistry and Biochemistry in Prague); i.e., acyclic nucleoside phosphonates (ANPs, e.g., adefovir, tenofovir, and cidofovir) and derivatives of non-nucleoside phosphonates such as 2-(phosphonomethyl) pentanedioic acid (2-PMPA). Principal strategies of their syntheses and modifications to prodrugs is reported. Besides clinically used ANP antivirals, a special attention is paid to new biologically active molecules with respect to emerging infections and arising resistance of many pathogens against standard treatments. These new structures include 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidines or so-called "open-ring" derivatives, acyclic nucleoside phosphonates with 5-azacytosine as a base moiety, side-chain fluorinated ANPs, aza/deazapurine ANPs. When transformed into an appropriate prodrug by derivatizing their charged functionalities, all these compounds show promising potential to become drug candidates for the treatment of viral infections. ANP prodrugs with suitable pharmacokinetics include amino acid phosphoramidates, pivaloyloxymethyl (POM) and isopropoxycarbonyloxymethyl (POC) esters, alkyl and alkoxyalkyl esters, salicylic esters, (methyl-2-oxo-1,3-dioxol-4-yl) methyl (ODOL) esters and peptidomimetic prodrugs. We also focus on the story of cytostatics related to 9-[2-(phosphonomethoxy)ethyl]guanine and its prodrugs which eventually led to development of the veterinary drug rabacfosadine. Various new ANP structures are also currently investigated as antiparasitics, especially antimalarial agents e.g., guanine and hypoxanthine derivatives with 2-(phosphonoethoxy)ethyl moiety, their thia-analogues and N-branched derivatives. In addition to ANPs and their analogs, we also describe prodrugs of 2-(phosphonomethyl)pentanedioic acid (2-PMPA), a potent inhibitor of the enzyme glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA). Glutamate carboxypeptidase II inhibitors, including 2-PMPA have been found efficacious in various preclinical models of neurological disorders which are caused by glutamatergic excitotoxicity. Unfortunately its highly polar character and hence low bioavailability severely limits its potential for clinical use. To overcome this problem, various prodrug strategies have been used to mask carboxylates and/or phosphonate functionalities with pivaloyloxymethyl, POC, ODOL and alkyl esters. Chemistry and biological characterization led to identification of prodrugs with 44-80 fold greater oral bioavailability (tetra-ODOL-2-PMPA).

• Keywords
• 2-PMPA, FOLH1, GCPII, acyclic nucleoside phosphonates, antivirals, prodrugs, prostate-specific membrane antigen, protides,
• Publication type
• Journal Article MeSH
• Review MeSH

All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).

• MeSH
• Adenine Phosphoribosyltransferase metabolism   MeSH
• Adenine Nucleotides metabolism   MeSH
• Cell Line MeSH
• HeLa Cells MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Nucleosides metabolism   MeSH
• Organophosphonates metabolism   MeSH
• Purines metabolism   MeSH
• Trypanosoma brucei brucei metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine Phosphoribosyltransferase MeSH
• Adenine Nucleotides MeSH
• Nucleosides MeSH
• Organophosphonates MeSH
• purine MeSH Browser
• Purines MeSH

A series of 13 acyclic nucleoside phosphonates (ANPs) as bisamidate prodrugs was prepared. Five compounds were found to be non-cytotoxic and selective inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT) in J774A.1 macrophage cell-based assays. The 8-aza-7-deazapurine derivative of adefovir (PMEA) was found to be the most potent ACT inhibitor in the series (IC50 =16 nm) with substantial selectivity over mammalian adenylate cyclases (mACs). AC inhibitory properties of the most potent analogues were confirmed by direct evaluation of the corresponding phosphonodiphosphates in cell-free assays and were found to be potent inhibitors of both ACT and edema factor (EF) from Bacillus anthracis (IC50 values ranging from 0.5 to 21 nm). Moreover, 7-halo-7-deazapurine analogues of PMEA were discovered to be potent and selective mammalian AC1 inhibitors (no inhibition of AC2 and AC5) with IC50 values ranging from 4.1 to 5.6 μm in HEK293 cell-based assays.

• Keywords
• Bacillus anthracis, Bordetella pertussis, adefovir, adenylate cyclase, inhibitors,
• MeSH
• Adenine analogs & derivatives   chemical synthesis   chemistry   pharmacology   MeSH
• Adenylyl Cyclases metabolism   MeSH
• Bacillus anthracis enzymology   MeSH
• Bordetella pertussis enzymology   MeSH
• Enzyme Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Molecular Structure MeSH
• Organophosphonates chemical synthesis   chemistry   pharmacology   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• adefovir MeSH Browser
• Adenine MeSH
• Adenylyl Cyclases MeSH
• Enzyme Inhibitors MeSH
• Organophosphonates MeSH

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites' viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.

• MeSH
• Hypoxanthine Phosphoribosyltransferase antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Enzyme Inhibitors chemistry   pharmacology   MeSH
• Catalytic Domain MeSH
• Humans MeSH
• Metabolic Networks and Pathways drug effects   MeSH
• Models, Molecular MeSH
• Drug Discovery MeSH
• Pentosyltransferases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Purinones metabolism   MeSH
• RNA Interference MeSH
• Trypanocidal Agents chemistry   pharmacology   MeSH
• Trypanosoma brucei brucei drug effects   enzymology   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• hypoxanthine-guanine-xanthine phosphoribosyltransferase MeSH Browser
• Hypoxanthine Phosphoribosyltransferase MeSH
• Enzyme Inhibitors MeSH
• Pentosyltransferases MeSH
• Purinones MeSH
• Trypanocidal Agents MeSH

Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF) are key virulence factors with adenylate cyclase (AC) activity that substantially contribute to the pathogenesis of whooping cough and anthrax, respectively. There is an urgent need to develop potent and selective inhibitors of bacterial ACs with prospects for the development of potential antibacterial therapeutics and to study their molecular interactions with the target enzymes. Novel fluorescent 5-chloroanthraniloyl-substituted acyclic nucleoside phosphonates (Cl-ANT-ANPs) were designed and synthesized in the form of their diphosphates (Cl-ANT-ANPpp) as competitive ACT and EF inhibitors with sub-micromolar potency (IC50 values: 11-622 nm). Fluorescence experiments indicated that Cl-ANT-ANPpp analogues bind to the ACT active site, and docking studies suggested that the Cl-ANT group interacts with Phe306 and Leu60. Interestingly, the increase in direct fluorescence with Cl-ANT-ANPpp having an ester linker was strictly calmodulin (CaM)-dependent, whereas Cl-ANT-ANPpp analogues with an amide linker, upon binding to ACT, increased the fluorescence even in the absence of CaM. Such a dependence of binding on structural modification could be exploited in the future design of potent inhibitors of bacterial ACs. Furthermore, one Cl-ANT-ANP in the form of a bisamidate prodrug was able to inhibit B. pertussis ACT activity in macrophage cells with IC50 =12 μm.

• Keywords
• adenylate cyclase, anthrax, antibacterial agents, fluorescence, whooping cough,
• MeSH
• Adenylyl Cyclases metabolism   MeSH
• Bordetella pertussis enzymology   MeSH
• Fluorescent Dyes chemical synthesis   chemistry   pharmacology   MeSH
• Adenylyl Cyclase Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Macrophages drug effects   MeSH
• Molecular Structure MeSH
• Mice MeSH
• Nucleosides chemical synthesis   chemistry   pharmacology   MeSH
• Organophosphonates chemical synthesis   chemistry   pharmacology   MeSH
• Drug Design * MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylyl Cyclases MeSH
• Fluorescent Dyes MeSH
• Adenylyl Cyclase Inhibitors MeSH
• Nucleosides MeSH
• Organophosphonates MeSH

Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that when two of the ANPs bind, they induce an unusual conformation change to the loop where the reaction product, pyrophosphate, is expected to bind. This and other structural differences between the Tbr and human enzymes suggest selective inhibitors for the Tbr enzyme can be designed.

• MeSH
• Species Specificity MeSH
• Hypoxanthine Phosphoribosyltransferase antagonists & inhibitors   chemistry   genetics   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Catalytic Domain MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protozoan Proteins antagonists & inhibitors   chemistry   genetics   MeSH
• Recombinant Proteins chemistry   genetics   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Trypanocidal Agents pharmacology   MeSH
• Trypanosoma brucei brucei drug effects   enzymology   genetics   MeSH
• Trypanosoma cruzi enzymology   genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hypoxanthine Phosphoribosyltransferase MeSH
• Enzyme Inhibitors MeSH
• Protozoan Proteins MeSH
• Recombinant Proteins MeSH
• Trypanocidal Agents MeSH