Petr Špaček

Inhibition of the Escherichia coli 6-oxopurine phosphoribosyltransferases by nucleoside phosphonates: potential for new antibacterial agents

Escherichia coli (Ec) cells possess two purine salvage enzymes: xanthine-guanine phosphoribosyltransferase (XGPRT) and hypoxanthine phosphoribosyltransferase (HPRT). EcXGPRT shares a common structural feature with other members of this family, a flexible loop that closes over the active site during catalysis. The replacement of six of these amino acids by alanine has no effect on the Km for the two substrates. However, the Ki for the nucleoside monophosphate increases by 27-fold, and the kcat is reduced by ∼200-fold. Nucleoside phosphonates (NP) are good inhibitors of EcXGPRT and EcHPRT, with Ki values as low as 10 nM. In the absence of the flexible loop, these values increase by 5- to 30-fold, indicating the importance of the loop for high-affinity inhibition. Crystal structures of two NPs in complex with EcXGPRT explain the tight binding. Prodrugs of NPs with low Ki values for EcXGPRT or EcHPRT exhibit IC50 values between 5 and 23 μM against Mycobacterium tuberculosis in cell-based assays, suggesting that these compounds are therapeutic leads against pathogenic bacteria.

First Crystal Structures of Mycobacterium tuberculosis 6-Oxopurine Phosphoribosyltransferase: Complexes with GMP and Pyrophosphate and with Acyclic Nucleoside Phosphonates Whose Prodrugs Have Antituberculosis Activity.

Human tuberculosis is a chronic infectious disease affecting millions of lives. Because of emerging resistance to current medications, new therapeutic drugs are needed. One potential new target is hypoxanthine-guanine phosphoribosyltransferase (MtHGPRT), a key enzyme of the purine salvage pathway. Here, newly synthesized acyclic nucleoside phosphonates (ANPs) have been shown to be competitive inhibitors of MtHGPRT with Ki values as low as 0.69 μM. Prodrugs of these compounds arrest the growth of a virulent strain of M. tuberculosis with MIC50 values as low as 4.5 μM and possess low cytotoxicity in mammalian cells (CC50 values as high as >300 μM). In addition, the first crystal structures of MtHGPRT (2.03-2.76 Å resolution) have been determined, three of these in complex with novel ANPs and one with GMP and pyrophosphate. These data provide a solid foundation for the further development of ANPs as selective inhibitors of MtHGPRT and as antituberculosis agents.

An efficient oxa-Michael addition to diethyl vinylphosphonate under mild reaction conditions

An oxa-Michael addition of various secondary and branched primary alcohols to diethyl vinylphosphonate was systematically studied and optimized. This efficient method precedes using of harsh reaction conditions (e.g. strong bases, high temperatures) and gains access to an important class of biologically active compounds in one step.

Synthesis and evaluation of symmetric acyclic nucleoside bisphosphonates as inhibitors of the Plasmodium falciparum, Plasmodium vivax and human 6-oxopurine phosphoribosyltransferases and the antimalarial activity of their prodrugs

Two new series of symmetric acyclic nucleoside bisphosphonates (ANbPs) have been synthesised as potential inhibitors of the Plasmodium falciparum (Pf) and vivax (Pv) 6-oxopurine phosphoribosyltransferases. The structural variability between these symmetric ANbPs lies in the number of atoms in the two acyclic linkers connecting the N9 atom of the purine base to each of two phosphonate groups and the branching point of the acyclic moiety relative to the purine base, which occurs at either the alpha or beta positions. Within each series, six different 6-oxopurine bases have been attached. In general, the ANbPs with either guanine or hypoxanthine have lower Ki values than for those containing either the 8-bromo or 7-deaza 6-oxopurine bases. The lowest Ki values obtained for the two parasite enzymes were 0.1μM (Pf) and 0.2μM (Pv) for this series of compounds. Two phosphoramidate prodrugs of these inhibitors exhibited antimalarial activity against Pf in infected erythrocyte cell culture with IC50 values of 0.8 and 1.5μM. These two compounds exhibited low cytotoxicity in human A549 cells having CC50 values of >300μM resulting in an excellent selectivity index.

Synthesis and Evaluation of Asymmetric Acyclic Nucleoside Bisphosphonates as Inhibitors of Plasmodium falciparum and Human Hypoxanthine–Guanine–(Xanthine) Phosphoribosyltransferase

Acyclic nucleoside bisphosphonates (ANbPs) have previously been shown to be good inhibitors of human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and Plasmodium falciparum (Pf) hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT). On the basis of this scaffold, a new series of ANbPs was synthesized. One of these new ANbPs, [3-(guanine-9-yl)-2-((2-phosphonoethoxy)methyl)propoxy]methylphosphonic acid, exhibited Ki values of 6 and 70 nM for human HGPRT and Pf HGXPRT, respectively. These low Ki values were achieved by inserting an extra carbon atom in the linker connecting the N9 atom of guanine to one of the phosphonate groups. The crystal structure of this ANbP in complex with human HGPRT was determined at 2.0 Å resolution and shows that it fills three key pockets in the active site. The most potent phosphoramidate prodrugs of these compounds have IC50 values in the low micromolar range in Pf lines and low toxicity in human A549 cells, demonstrating that these ANbPs are excellent antimalarial drug leads.

Acyclic nucleoside phosphonates containing a second phosphonate group are potent inhibitors of the 6-oxopurine phosphoribosyltransferases and have antimalarial activity

Background The 6-oxopurine phosphoribosyltransferases have been suggested to be a target for the discovery of new antimalarial drugs. This is because protozoan parasites rely solely on the salvage of purines from their host to make the nucleotides needed for RNA and DNA synthesis and lack the de novo pathway. Acyclic nucleoside phosphonates (ANPs) that contain a 6-oxopurine base are good inhibitors of the Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) 6-oxopurine phosphoribosyltransferases (PRTs) [1]. Chemical modifications based on the crystal structure of 2-(phosphonoethoxy) ethylguanine (PEEG) in complex with human HGPRT have led to the design of new ANPs [2]. These novel compounds contain a second phosphonate group attached to the ANP scaffold [3].