Jikun Li

Bioorganometallic mechanism of action, and inhibition, of IspH

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 1H, 2H, 13C, 31P, and 57Fe-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 57Fe 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+ 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 1H, 2H, and 13C ENDOR, where hyperfine couplings of approximately 6 MHz for 13C and 10 MHz for 1H, 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 Fe4S4-containing proteins, such as IspG.

Organometallic mechanism of action and inhibition of the 4Fe-4S isoprenoid biosynthesis protein GcpE (IspG)

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 Fe4S4 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 2H, 13C and 17O 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 η2-alkenyl π- (or π/σ) complex with the 4th Fe in the Fe4S4 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 (Ki ∼ 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a 13C hyperfine coupling of ∼7 MHz), as well as literature precedent, we propose that the alkyne forms another π/σ metallacycle, an η2-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.

Lipophilic Pyridinium Bisphosphonates: Potent γδ T Cell Stimulators

Bisphosphonates such as risedronate and ibandronate are widely used to treat a variety of bone resorption diseases, preventing protein prenylation and disrupting osteoclast function. Bisphosphonates also activate human gd T cells (expressing the Vg2Vd2 T cell receptor), and these activated gd T cells kill tumor cells. 3] There has thus been interest in using bisphosphonates in cancer immunotherapy, with promising results against B-cell malignancies and hormone refractory prostate cancer. In a very recent clinical trial, it was shown that zoledronate offered a significant anticancer benefit when added to hormone therapy, reducing the risk of cancer returning by 36%. The bisphosphonates used in these trials are, however, extremely polar and are rapidly removed from circulation by binding to bone. We reasoned that it might be possible to develop more lipophilic bisphosphonates as gd T cell stimulators that would have improved cell uptake properties as well as decreased bone binding affinity. Herein, we report that novel lipophilic pyridinium bisphosphonates are approximately 250 times more effective in gd T cell activation than any other bisphosphonate drugs. Current nitrogen-containing bisphosphonates are thought to act primarily by blocking farnesyl diphosphate (FPP) formation in the isoprene biosynthesis pathway (Figure 1),

Non-Bisphosphonate Inhibitors of Isoprenoid Biosynthesis Identified via Computer-Aided Drug Design

The relaxed complex scheme, a virtual‐screening methodology that accounts for protein receptor flexibility, was used to identify a low‐micromolar, non‐bisphosphonate inhibitor of farnesyl diphosphate synthase. Serendipitously, we also found that several predicted farnesyl diphosphate synthase inhibitors were low‐micromolar inhibitors of undecaprenyl diphosphate synthase. These results are of interest because farnesyl diphosphate synthase inhibitors are being pursued as both anti‐infective and anticancer agents, and undecaprenyl diphosphate synthase inhibitors are antibacterial drug leads.

Pyridine Inhibitor Binding to the 4Fe-4S Protein A. aeolicus IspH (LytB): A HYSCORE Investigation

IspH is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using hyperfine sublevel correlation (HYSCORE) spectroscopy, we show that pyridine inhibitors of IspH directly bind to the unique fourth Fe in the 4Fe-4S cluster, opening up new routes to inhibitor design, of interest in the context of both anti-bacterial as well as anti-malarial drug discovery.

Squalene synthase as a target for Chagas disease therapeutics.

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.

Structure, function and inhibition of the two- and three-domain 4Fe-4S IspG proteins

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.

An ENDOR and HYSCORE Investigation of a Reaction Intermediate in IspG (GcpE) Catalysis

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 have specifically assigned the hyperfine interactions in a reaction intermediate. These results help clarify the nature of the reaction intermediate, supporting a direct interaction between the unique fourth Fe in the cluster and C2 and O3 of the ligand.

Lipophilic pyridinium bisphosphonates: potent gammadelta T cell stimulators.

Bisphosphonates such as risedronate and ibandronate are widely used to treat a variety of bone resorption diseases, preventing protein prenylation and disrupting osteoclast function. Bisphosphonates also activate human gd T cells (expressing the Vg2Vd2 T cell receptor), and these activated gd T cells kill tumor cells. 3] There has thus been interest in using bisphosphonates in cancer immunotherapy, with promising results against B-cell malignancies and hormone refractory prostate cancer. In a very recent clinical trial, it was shown that zoledronate offered a significant anticancer benefit when added to hormone therapy, reducing the risk of cancer returning by 36%. The bisphosphonates used in these trials are, however, extremely polar and are rapidly removed from circulation by binding to bone. We reasoned that it might be possible to develop more lipophilic bisphosphonates as gd T cell stimulators that would have improved cell uptake properties as well as decreased bone binding affinity. Herein, we report that novel lipophilic pyridinium bisphosphonates are approximately 250 times more effective in gd T cell activation than any other bisphosphonate drugs. Current nitrogen-containing bisphosphonates are thought to act primarily by blocking farnesyl diphosphate (FPP) formation in the isoprene biosynthesis pathway (Figure 1),

Isoprenoid Biosynthesis: Ferraoxetane or Allyl Anion Mechanism for IspH Catalysis?†

There are ~ 65,000 terpenes known.[1] These molecules are produced from two isoprenoid (C5) diphosphates: isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2).[2] In plant plastids, IPP and DMAPP are produced primarily via the 2-C-methylerythritol 4-phosphate (MEP) pathway from E-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP, 3) in a reaction catalyzed by the enzyme IspH, and this route is also used in most bacteria, as well as in malaria parasites.