Christoph Behrendt

Reverse Fosmidomycin Derivatives against the Antimalarial Drug Target IspC (Dxr).

Reverse hydroxamate-based inhibitors of IspC, a key enzyme of the non-mevalonate pathway of isoprenoid biosynthesis and a validated antimalarial target, were synthesized and biologically evaluated. The binding mode of one derivative in complex with EcIspC and a divalent metal ion was clarified by X-ray analysis. Pilot experiments have demonstrated in vivo potential.

Synthesis and Antiplasmodial Activity of Highly Active Reverse Analogues of the Antimalarial Drug Candidate Fosmidomycin

Although there are potent antimalarial drugs available, the spread of drug resistance has led to an increase in morbidity and mortality rates in malaria-endemic regions. To overcome these problems, new antimalarial drugs with novel modes of action have to be developed. The recently discovered nonmevalonate pathway of isoprenoid biosynthesis (MEP pathway) is an important metabolic pathway for the design of novel antimalarial drugs. Various pathogenic bacteria (e.g. , Mycobacterium tuberculosis) and apicomplexan protozoa (e.g. , Plasmodium falciparum) use the MEP pathway for the production of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP are important precursors for the biosynthesis of various essential isoprenoids. In contrast to the latter microorganisms, humans produce IPP and DMAPP exclusively via the mevalonate–acetate pathway. The second enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) represents a validated target enzyme for the development of new antimalarial drugs. In 1998, the natural product fosmidomycin was identified as a potent inhibitor of P. falciparum DXR (PfDXR). The DXR catalyzes the rearrangement and reduction of 1-deoxy-d-xylulose 5-phosphate (DOXP) into 2-C-methyl-d-erythritol 4-phosphate (MEP). While the first step of the reaction requires a divalent cation (e.g. , Mn + or Mg ), the reduction step is NADPH-dependent. Clinical trials performed with fosmidomycin have already shown high efficiency in the treatment of acute, uncomplicated malaria tropica. However, the oral bioavailability of fosmidomycin is only moderate, with a resorption rate of about 10–30 %. 8] Various research groups are involved in the design of novel DXR inhibitors. Molecular field analyses, as well as crystallographic studies, could already contribute in defining structural requirements for the development of potent DXR inhibitors. 10] Consequently, different types of DXR inhibitors have been synthesized and biologically evaluated. 4, 11] Important structural elements for potent antimalarial activity are the hydroxamic acid functionality and the phosphonic acid group. 11] The DXR–fosmidomycin complex crystal structure clearly revealed that the hydroxamic acid functionality chelates the divalent cation, while the phosphonic acid group occupies the phosphate binding site. Moreover, a defined distance between both functional groups is essential for potent antimalarial activity. Schlitzer and Klebe have shown that the two negative charges of the phosphonic acid group are necessary for high inhibitory activity. Furthermore, phosphonate prodrugs have been designed to improve the bioavailability of fosmidomycin (1) and FR900098 (2). A combinatorial approach for the synthesis of nonhydrolyzable phosphate mimics was reported by Link and co-workers, while Song recently described a coordination chemistry-based approach for the development of lipophilic DXR inhibitors that exhibit good to moderate activity against various pathogenic bacteria. To the best of our knowledge, a-aryl-substituted derivatives (4) of fosmidomycin are among the most active analogues known so far. Recently, Van Calenbergh reported that b-oxaisosters (3 a,b) of fosmidomycin exhibit strong in vitro antimalarial activity. Interestingly, some of the new compounds contain an N-methyl-substituted hydroxamic acid group with a reverse orientation of the hydroxamate group (3 b) compared to the lead compounds (1 and 2). Due to difficulties associated with the handling of PfDXR, no data concerning the inhibitory activity of these analogues toward PfDXR have been reported. 17] Furthermore, Rohmer and co-workers presented several reverse fosmidomycin analogues, which in part inhibit recombinant E. coli DXR (EcDXR). In order to improve the antiplasmodial activity and [a] C. T. Behrendt, Prof. Dr. T. Kurz Institut f r Pharmazeutische und Medizinische Chemie Heinrich-Heine-Universit t D sseldorf Universit tsstr. 1, 40225 D sseldorf (Germany) Fax: (+ 49) 211-811-3847 E-mail : Thomas.Kurz@uni-duesseldorf.de [b] A. Kunfermann, Dr. T. Gr wert, Prof. M. Groll, Dr. F. Rohdich, Prof. A. Bacher, Dr. W. Eisenreich Lehrstuhl f r Biochemie, Center for Integrated Protein Science M nchen Technische Universit t M nchen Lichtenbergstr. 4, 85747 Garching (Germany) [c] Dr. V. Illarionova, Prof. Dr. M. Fischer Institut f r Lebensmittelchemie, Universit t Hamburg Grindelallee 117, 20146 Hamburg (Germany) [d] A. Matheeussen, Prof. Dr. L. Maes Laboratory for Microbiology, Parasitology and Hygiene (LMPH) University of Antwerp, Groenenborgerlaan 171, 2020 Wilrijk (Belgium) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000276.

Binding modes of reverse fosmidomycin analogs toward the antimalarial target IspC.

1-Deoxy-d-xylulose 5-phosphate reductoisomerase of Plasmodium falciparum (PfIspC, PfDxr), believed to be the rate-limiting enzyme of the nonmevalonate pathway of isoprenoid biosynthesis (MEP pathway), is a clinically validated antimalarial target. The enzyme is efficiently inhibited by the natural product fosmidomycin. To gain new insights into the structure activity relationships of reverse fosmidomycin analogs, several reverse analogs of fosmidomycin were synthesized and biologically evaluated. The 4-methoxyphenyl substituted derivative 2c showed potent inhibition of PfIspC as well as of P. falciparum growth and was more than one order of magnitude more active than fosmidomycin. The binding modes of three new derivatives in complex with PfIspC, reduced nicotinamide adenine dinucleotide phosphate, and Mg(2+) were determined by X-ray structure analysis. Notably, PfIspC selectively binds the S-enantiomers of the study compounds.

α-Phenylethyl Substituted Bis(pivaloyloxymethyl) Ester Analogues of Fosmidomycin and FR900098

α-Phenylethyl substituted bis(pivaloyloxymethyl) ester analogues of the natural products Fosmidomycin and FR900098 have been synthesized, and their in vitro antimalarial activity determined. The α-phenylethyl substituted Fosmidomycin analogue displays moderate in vitro antimalarial activity against the chloroquine-sensitive strain 3D7 of Plasmodium falciparum.

Crystal Structure of Brucella abortus Deoxyxylulose-5-phosphate Reductoisomerase-like (DRL) Enzyme Involved in Isoprenoid Biosynthesis

Background: The current antibiotic resistance epidemic demands new drugs specifically targeting infective agents. Results: The crystal structure of the Brucella DRL enzyme shows major differences compared with DXR, which catalyzes the same reaction in most other bacteria. Conclusion: Structural information will allow development of inhibitors targeting only DRL. Significance: Drugs against DRL could function as highly specific, narrow-range antibiotics. Most bacteria use the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for the synthesis of their essential isoprenoid precursors. The absence of the MEP pathway in humans makes it a promising new target for the development of much needed new and safe antimicrobial drugs. However, bacteria show a remarkable metabolic plasticity for isoprenoid production. For example, the NADPH-dependent production of MEP from 1-deoxy-d-xylulose 5-phosphate in the first committed step of the MEP pathway is catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in most bacteria, whereas an unrelated DXR-like (DRL) protein was recently found to catalyze the same reaction in some organisms, including the emerging human and animal pathogens Bartonella and Brucella. Here, we report the x-ray crystal structures of the Brucella abortus DRL enzyme in its apo form and in complex with the broad-spectrum antibiotic fosmidomycin solved to 1.5 and 1.8 Å resolution, respectively. DRL is a dimer, with each polypeptide folding into three distinct domains starting with the NADPH-binding domain, in resemblance to the structure of bacterial DXR enzymes. Other than that, DRL and DXR show a low structural relationship, with a different disposition of the domains and a topologically unrelated C-terminal domain. In particular, the active site of DRL presents a unique arrangement, suggesting that the design of drugs that would selectively inhibit DRL-harboring pathogens without affecting beneficial or innocuous bacteria harboring DXR should be feasible. As a proof of concept, we identified two strong DXR inhibitors that have virtually no effect on DRL activity.

Prodrugs of reverse fosmidomycin analogues

Fosmidomycin inhibits IspC (Dxr, 1-deoxy-d-xylulose 5-phosphate reductoisomerase), a key enzyme in nonmevalonate isoprenoid biosynthesis that is essential in Plasmodium falciparum. The drug has been used successfully to treat malaria patients in clinical studies, thus validating IspC as an antimalarial target. However, improvement of the drugs pharmacodynamics and pharmacokinetics is desirable. Here, we show that the conversion of the phosphonate moiety into acyloxymethyl and alkoxycarbonyloxymethyl groups can increase the in vitro activity against asexual blood stages of P. falciparum by more than 1 order of magnitude. We also synthesized double prodrugs by additional esterification of the hydroxamate moiety. Prodrugs with modified hydroxamate moieties are subject to bioactivation in vitro. All prodrugs demonstrated improved antiplasmodial in vitro activity. Selected prodrugs and parent compounds were also tested for their cytotoxicity toward HeLa cells and in vivo in a Plasmodium berghei malaria model as well as in the SCID mouse P. falciparum model.