Inhibition of the 4Fe–4S proteins IspG and IspH: an EPR, ENDOR and HYSCORE investigation

Abstract

IspG and IspH are proteins that are involved in isoprenoid biosynthesis in most bacteria as well as in malaria parasites and are important drug targets. They contain cubane-type 4Fe-4S clusters that are involved in unusual 2H+/2e− reductions. Here, we report the results of electron paramagnetic resonance spectroscopic investigations of the binding of amino- and thiolo-HMBPP (HMBPP=E-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate) IspH substrate-analog inhibitors to both proteins, as well as the binding of HMBPP and an acetylene diphosphate inhibitor, to IspG. The results show that amino-HMBPP binds to reduced IspH by Fe-C π-bonding with the olefinic carbons interacting with the unique 4th Fe in the 4Fe-4S cluster, quite different to the direct Fe-N ligation seen with the oxidized protein. No such π-complex is observed when amino-HMBPP binds to reduced IspG. No EPR signal is observed with IspH in the presence of dithionite and thiolo-HMBPP, suggesting that the 4Fe-4S cluster is not reduced, consistent with the presence of a 420 nm feature in the absorption spectrum (characteristic of an oxidized cluster). However, with IspG, the EPR spectrum in the presence of dithionite and thiolo-HMBPP is very similar to that seen with HMBPP. The binding of HMBPP to IspG was studied using hyperfine sublevel correlation spectroscopy with 17O and 13C labeled samples: the results rule out direct Fe-O bonding and indicate π-bonding. Finally, the binding to IspG of a potent acetylene diphosphate inhibitor was studied by using electron-nuclear double resonance spectroscopy with 13C labeled ligands: the large hyperfine couplings indicate strong Fe-C π-bonding with the acetylenic group. These results illustrate a remarkable diversity in binding behavior for HMBPP-analog inhibitors, opening up new routes to inhibitor design of interest in the context of anti-bacterial and anti-malarial drug discovery, as well as “cubane-type” metallo-biochemistry, in general.

Atomic-Resolution Structures of Discrete Stages on the Reaction Coordinate of the [Fe 4 S 4 ] Enzyme IspG (GcpE)

“…In response, a variety of diphosphate-containing inhibitors have been designed and screened against the enzyme [4, 13, 17-19]. Several of them were shown to exhibit activity against both IspG as well as IspH, the downstream catalyst and final component of the MEP pathway [18, 20]. In addition to the identification of the reaction intermediates described above, minimal requirements for ligand binding are essential pieces of information for the design and improvement of such inhibitors.…”

New Insight into Isoprenoids Biosynthesis Process and Future Prospects for Drug Designing in Plasmodium

“…Bhuyan et al ( 2015 ) screened a large ligand data-set containing diphospate group against the P. falciparum IspH structure, and based on Goldscore and Chemscore identified 17 lead compounds amongst which 5-((hydroxymethyl)-O-pyrophosphoryl) uracil shows best binding affinities with Plasmodium IspH, and thus can be considered as its potential inhibitor. Recently, the derivatives of diphosphonate such as alkyl phosphate have been identified as potential inhibitors for IspG and IspH enzymes of A. aeolicus, E. coli , and P. falciparum (Guerra et al, 2014 ), however their exact mechanism for inhibition is unknown.…”

IspH–RPS1 and IspH–UbiA: “Rosetta stone” proteins

“…The methylerythritol phosphate pathway is responsible for the production of the terpene building blocks dimethylallyl diphosphate (DMAPP, 1 , Scheme 1 ) and isopentenyl diphosphate (IPP, 2 ) in most bacteria, apicomplexan parasites, and plants. Its component enzymes are all essential for survival, so there is interest in their inhibition 1 , 2 for the development of drugs to treat infectious diseases, such as malaria and tuberculosis, with fosmidomycin, 3 which inhibits deoxyxylulose 4-phosphate reductoisomerase, having reached clinical trials for malaria. 4 In addition, targeting the pathway could be of use for herbicide development.…”