We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M ; V max ), EPR and 1 H, 2 H, 13 C, 31 P, and 57 Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on 57 Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π∕σ "metallacycle" or η 2 -alkenyl complex. The complex is poised to interact with H þ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η 1 -allyl complex. After reduction, this forms an η 3 -allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by 1 H, 2 H, and 13 C ENDOR, where hyperfine couplings of approximately 6 MHz for 13 C and 10 MHz for 1 H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe 4 S 4 -containing proteins, such as IspG.enzyme inhibition | iron-sulfur protein | isoprenoid biosynthesis | nonmevalonate pathway E nzymes that catalyze the formation of isoprenoids are of interest as drug targets. There are two main pathways involved in the early steps in isoprenoid biosynthesis: The mevalonate pathway found in animals and in pathogens such as Staphylococcus aureus, Trypanosoma cruzi, and Leishmania spp. (the causative agents of staph infections, Chagas' disease and the leishmaniases), and the nonmevalonate or Rohmer pathway found in most pathogenic bacteria, as well as in the malaria parasite, Plasmodium falciparum (1). Both pathways lead to formation of the C 5 -isoprenoids isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2). In the later stages of isoprenoid biosynthesis, these C 5 -compounds then form the farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) used in protein prenylation, sterol, and carotenoid biosynthesis. Understanding how the enzymes catalyzing these "downstream" events function has led to a better understanding of e.g. how FPP synthase (2) and GGPP synthase function, and can be inhibited (3); the discovery that bisphosphonates have potent antiparasitic activity (4); the clinical use of amiodarone (a squalene oxidase and oxidosqualene cyclase inhibitor) against Chagas' disease (5; 6) and leishmaniasis (7); anticancer agents that inhibit both FPPS and GGPPS (8); as wel...

There is a growing need for new antibiotics. Compounds that target the proton motive force (PMF), uncouplers, represent one possible class of compounds that might be developed because they are already used to treat parasitic infections, and there is interest in their use for the treatment of other diseases, such as diabetes. Here, we tested a series of compounds, most with known antiinfective activity, for uncoupler activity. Many cationic amphiphiles tested positive, and some targeted isoprenoid biosynthesis or affected lipid bilayer structure. As an example, we found that clomiphene, a recently discovered undecaprenyl diphosphate synthase inhibitor active against Staphylococcus aureus, is an uncoupler. Using in silico screening, we then found that the anti-glioblastoma multiforme drug lead vacquinol is an inhibitor of Mycobacterium tuberculosis tuberculosinyl adenosine synthase, as well as being an uncoupler. Because vacquinol is also an inhibitor of M. tuberculosis cell growth, we used similarity searches based on the vacquinol structure, finding analogs with potent (∼0.5-2 μg/mL) activity against M. tuberculosis and S. aureus. Our results give a logical explanation of the observation that most new tuberculosis drug leads discovered by phenotypic screens and genome sequencing are highly lipophilic (logP ∼5.7) bases with membrane targets because such species are expected to partition into hydrophobic membranes, inhibiting membrane proteins, in addition to collapsing the PMF. This multiple targeting is expected to be of importance in overcoming the development of drug resistance because targeting membrane physical properties is expected to be less susceptible to the development of resistance.T here is a need for new antibiotics, due to the increase in drug resistance (1, 2). For example, some studies report that by 2050, absent major improvements in drug discovery and use, more individuals will die from drug-resistant bacterial infections than from cancer, resulting in a cumulative effect on global gross domestic product of as much as 100 trillion dollars (3, 4). To discover new drugs, new targets, leads, concepts, and implementations are needed (5, 6).Currently, one major cause of death from bacterial infections is tuberculosis (TB) (7), where very highly drug-resistant strains have been found (8). Therapy is lengthy, even with drug-sensitive strains, and requires combination therapies with four drugs. Two recent TB drugs/drug leads (9-11) are TMC207 [bedaquiline (1); Sirturo] and SQ109 (2) (Fig. 1). Bedaquiline (1) targets the Mycobacterium tuberculosis ATP synthase (9) whereas SQ109 (2) has been proposed to target MmpL3 (mycobacterial membrane protein large 3), a trehalose monomycolate transporter essential for cell wall biosynthesis (12). SQ109 (2) is a lipophilic base containing an adamantyl "headgroup" connected via an ethylene diamine "linker" to a geranyl (C 10 ) "side chain," and in recent work (13), we synthesized a series of 11 analogs of SQ109 (2) finding that the ethanolamine (3) was more potent th...

We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe 4 S 4 cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using 2 H, 13 C and 17 O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an η 2 -alkenyl π-(or π∕σ) complex with the 4th Fe in the Fe 4 S 4 cluster, and a similar "metallacycle" also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (K i ∼ 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a 13 C hyperfine coupling of ∼7 MHz), as well as literature precedent, we propose that the alkyne forms another π∕σ metallacycle, an η 2 -alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role.4Fe-4S protein | GcpE (IspG) | metallacycle

The development of helicene molecules with significant chiroptical responses covering a broad range of the visible spectrum is highly desirable for chiral optoelectronic applications; however, their absorption dissymmetry factors (g abs ) have been mostly lower than 0.01. In this work, we report unprecedented B,N-embedded double hetero[7]helicenes with nonbonded B and N atoms, which exhibit excellent chiroptical properties, such as strong chiroptical activities from 300 to 700 nm, record high g abs up to 0.033 in the visible spectral range, and tunable circularly polarized luminescence (CPL) from red to near-infrared regions (600−800 nm) with high photoluminescence quantum yields (PLQYs) up to 100%. As revealed by theoretical analyses, the high g abs values are related to the separate molecular orbital distributions owing to the incorporation of nonbonded B and N atoms. The new type of B,N-embedded double heterohelicenes opens up an appealing avenue to the future exploitation of high-performance chiroptical materials.

IspG is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies on labeled samples, we specifically assign the hyperfine interactions in a reaction intermediate. These results help clarify the nature of the reaction intermediate, supporting a direct interaction between the unique 4th Fe in the cluster and the C2 and O3 of the ligand.

High-valent nonheme Fe IV −oxido species are key intermediates in biological oxidation, and their properties are proposed to be influenced by the unique microenvironments present in protein active sites. Microenvironments are regulated by noncovalent interactions, such as hydrogen bonds (H-bonds) and electrostatic interactions; however, there is little quantitative information about how these interactions affect crucial properties of high valent metal−oxido complexes. To address this knowledge gap, we introduced a series of Fe IV −oxido complexes that have the same S = 2 spin ground state as those found in nature and then systematically probed the effects of noncovalent interactions on their electronic, structural, and vibrational properties. The key design feature that provides access to these complexes is the new tripodal ligand [poat] 3− , which contains phosphinic amido groups. An important structural aspect of [Fe IV poat(O)] − is the inclusion of an auxiliary site capable of binding a Lewis acid (LA II ); we used this unique feature to further modulate the electrostatic environment around the Fe−oxido unit. Experimentally, studies confirmed that H-bonds and LA II s can interact directly with the oxido ligand in Fe IV −oxido complexes, which weakens the FeO bond and has an impact on the electronic structure. We found that relatively large vibrational changes in the Fe−oxido unit correlate with small structural changes that could be difficult to measure, especially within a protein active site. Our work demonstrates the important role of noncovalent interactions on the properties of metal complexes, and that these interactions need to be considered when developing effective oxidants.

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

IspG is a 4Fe4S protein involved in isoprenoid biosynthesis. Most bacterial IspGs contain two domains: a TIM barrel (A) and a 4Fe4S domain (B), but in plants and malaria parasites, there is a large insert domain (A*) whose structure and function are unknown. We show that bacterial IspGs function in solution as (AB) 2 dimers and that mutations in either both A or both B domains block activity. Chimeras harboring an A-mutation in one chain and a B-mutation in the other have 50% of the activity seen in wild-type protein, because there is still one catalytically active AB domain. However, a plant IspG functions as an AA*B monomer. We propose, using computational modeling and electron microscopy, that the A* insert domain has a TIM barrel structure that interacts with the A domain. This structural arrangement enables the A and B domains to interact in a “cup and ball” manner during catalysis, just as in the bacterial systems. EPR/HYSCORE spectra of reaction intermediate, product, and inhibitor ligands bound to both two and three domain proteins are identical, indicating the same local electronic structure, and computational docking indicates these ligands bridge both A and B domains. Overall, the results are of broad general interest because they indicate the insert domain in three-domain IspGs is a second TIM barrel that plays a structural role and that the pattern of inhibition of both two and three domain proteins are the same, results that can be expected to be of use in drug design.