Terpenes are the largest class of small‐molecule natural products on earth, and the most abundant by mass. Here, we summarize recent developments in elucidating the structure and function of the ...proteins involved in their biosynthesis. There are six main building blocks or modules (α, β, γ, δ, ε, and ζ) that make up the structures of these enzymes: the αα and αδ head‐to‐tail trans‐prenyl transferases that produce trans‐isoprenoid diphosphates from C5 precursors; the ε head‐to‐head prenyl transferases that convert these diphosphates into the tri‐ and tetraterpene precursors of sterols, hopanoids, and carotenoids; the βγ di‐ and triterpene synthases; the ζ head‐to‐tail cis‐prenyl transferases that produce the cis‐isoprenoid diphosphates involved in bacterial cell wall biosynthesis; and finally the α, αβ, and αβγ terpene synthases that produce plant terpenes, with many of these modular enzymes having originated from ancestral α and β domain proteins. We also review progress in determining the structure and function of the two 4Fe‐4S reductases involved in formation of the C5 diphosphates in many bacteria, where again, highly modular structures are found.
Natural building blocks: Recent progress has been achieved in determining the structure, function, and inhibition of the enzymes responsible for the formation of terpenes and isoprenoids. Most of these enzymes contain combinations of α‐, β‐, γ‐, δ‐, ε‐, and/or ζ‐domain structures that in many cases are fused to form modular proteins. Gene fusion, exon loss, and recombination are thought to play major roles in the genesis of these enzymes.
We show that copper-containing metal–organic nanoparticles (MONPs) are readily synthesized via Cu(II)-mediated intramolecular cross-linking of aspartate-containing polyolefins in water. In situ ...reduction with sodium ascorbate yields Cu(I)-containing MONPs that serve as highly efficient supramolecular catalysts for alkyne–azide “click chemistry” reactions, yielding the desired 1,4-adducts at low parts per million catalyst levels. The nanoparticles have low toxicity and low metal loadings, making them convenient, green catalysts for alkyne–azide “click” reactions in water. The Cu-MONPs enter cells and perform efficient, biocompatible click chemistry, thus acting as intracellular nanoscale molecular synthesizers.
Motivated by the clinical observation that interruption of the mevalonate pathway stimulates immune responses, we hypothesized that this pathway may function as a druggable target for vaccine ...adjuvant discovery. We found that lipophilic statin drugs and rationally designed bisphosphonates that target three distinct enzymes in the mevalonate pathway have potent adjuvant activities in mice and cynomolgus monkeys. These inhibitors function independently of conventional “danger sensing.” Instead, they inhibit the geranylgeranylation of small GTPases, including Rab5 in antigen-presenting cells, resulting in arrested endosomal maturation, prolonged antigen retention, enhanced antigen presentation, and T cell activation. Additionally, inhibiting the mevalonate pathway enhances antigen-specific anti-tumor immunity, inducing both Th1 and cytolytic T cell responses. As demonstrated in multiple mouse cancer models, the mevalonate pathway inhibitors are robust for cancer vaccinations and synergize with anti-PD-1 antibodies. Our research thus defines the mevalonate pathway as a druggable target for vaccine adjuvants and cancer immunotherapies.
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•Lipophilic statins and lipophilic bisphosphonates are potent vaccine adjuvants•Modulation of post-translational protein prenylation confers adjuvanticity•Decreased protein prenylation augments antigen preservation and presentation•Statin- or bisphosphonate-mediated vaccination synergizes with anti-PD1 against cancer
Lipophilic statins and bisphosphonates that target three enzymes in the mevalonate pathway have vaccine adjuvant activities in mice and monkeys and can synergize with anti-PD1 therapy.
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 againstStaphylococcus aureus, is an uncoupler. Using in silico screening, we then found that the anti-glioblastoma multiforme drug lead vacquinol is an inhibitor ofMycobacterium tuberculosistuberculosinyl adenosine synthase, as well as being an uncoupler. Because vacquinol is also an inhibitor ofM. tuberculosiscell growth, we used similarity searches based on the vacquinol structure, finding analogs with potent (∼0.5–2 μg/mL) activity againstM. tuberculosisandS. 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.
We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite. The compounds are ...analogues of the new tuberculosis (TB) drug SQ109 (1), which has been reported to act by inhibiting a transporter called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone biosynthesis and electron transport, inhibiting respiration and ATP biosynthesis, and are uncouplers, collapsing the pH gradient and membrane potential used to power transporters. The result of such multitarget inhibition is potent inhibition of TB cell growth, as well as very low rates of spontaneous drug resistance. Several targets are absent in humans but are present in other bacteria, as well as in malaria parasites, whose growth is also inhibited.
Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, ...antagonistic interactions can also have tremendous value in elucidating genetic networks andmechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase inStaphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis inS. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.
The isoprenoid biosynthesis pathways produce the largest class of small molecules in Nature: isoprenoids (also called terpenoids). Not surprisingly then, isoprenoid biosynthesis is a target for drug ...discovery, and many drugssuch as Lipitor (used to lower cholesterol), Fosamax (used to treat osteoporosis), and many anti-infectivestarget isoprenoid biosynthesis. However, drug resistance in malaria, tuberculosis, and staph infections is rising, cheap and effective drugs for the neglected tropical diseases are lacking, and progress in the development of anticancer drugs is relatively slow. Isoprenoid biosynthesis is thus an attractive target, and in this Account, I describe developments in four areas, using in each case knowledge derived from one area of chemistry to guide the development of inhibitors (or drug leads) in another, seemingly unrelated, area. First, I describe mechanistic studies of the enzyme IspH, which is present in malaria parasites and most pathogenic bacteria, but not in humans. IspH is a 4Fe−4S protein and produces the five-carbon (C5) isoprenoids IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) from HMBPP (E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate) via a 2H+/2e− reduction (of an allyl alcohol to an alkene). The mechanism is unusual in that it involves organometallic species: “metallacycles” (η2-alkenes) and η1/η3-allyls. These observations lead to novel alkyne inhibitors, which also form metallacycles. Second, I describe structure−function−inhibition studies of FPP synthase, the macromolecule that condenses IPP and DMAPP to the sesquiterpene farnesyl diphosphate (FPP) in a “head-to-tail” manner. This enzyme uses a carbocation mechanism and is potently inhibited by bone resorption drugs (bisphosphonates), which I show are also antiparasitic agents that block sterol biosynthesis in protozoa. Moreover, “lipophilic” bisphosphonates inhibit protein prenylation and invasiveness in tumor cells, in addition to activating γδ T-cells to kill tumor cells, and are important new leads in oncology. Third, I describe structural and inhibition studies of a “head-to-head” triterpene synthase, dehydrosqualene synthase (CrtM), from Staphylococcus aureus. CrtM catalyzes the first committed step in biosynthesis of the carotenoid virulence factor staphyloxanthin: the condensation of two FPP molecules to produce a cyclopropane (presqualene diphosphate). The structure of CrtM is similar to that of human squalene synthase (SQS), and some SQS inhibitors (originally developed as cholesterol-lowering drugs) block staphyloxanthin biosynthesis. Treated bacteria are white and nonvirulent (because they lack the carotenoid shield that protects them from reactive oxygen species produced by neutrophils), rendering them susceptible to innate immune system clearancea new therapeutic approach. And finally, I show that the heart drug amiodarone, also known to have antifungal activity, blocks ergosterol biosynthesis at the level of oxidosqualene cyclase in Trypanosoma cruzi, work that has led to its use in the clinic as a novel antiparasitic agent. In each of these four examples, I use information from one area (organometallic chemistry, bone resorption drugs, cholesterol-lowering agents, heart disease) to develop drug leads in an unrelated area: a “knowledge-based” approach that represents an important advance in the search for new drugs.
The emergence of antibiotic-resistant bacterial species, such as vancomycin-resistant enterococci (VRE), necessitates the development of new antimicrobials. Here, we investigate the spectrum of ...antibacterial activity of three phenylthiazole-substituted aminoguanidines. These compounds possess potent activity against VRE, inhibiting growth of clinical isolates at concentrations as low as 0.5 μg/mL. The compounds exerted a rapid bactericidal effect, targeting cell wall synthesis. Transposon mutagenesis suggested three possible targets: YubA, YubB (undecaprenyl diphosphate phosphatase (UPPP)), and YubD. Both UPPP as well as undecaprenyl diphosphate synthase were inhibited by compound 1. YubA and YubD are annotated as transporters and may also be targets because 1 collapsed the proton motive force in membrane vesicles. Using Caenorhabditis elegans, we demonstrate that two compounds (1, 3, at 20 μg/mL) retain potent activity in vivo, significantly reducing the burden of VRE in infected worms. Taken altogether, the results indicate that compounds 1 and 3 warrant further investigation as novel antibacterial agents against drug-resistant enterococci.
Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of P i residues) of short chain polyP were found. The presence of polyP of â¼70â75 ...phosphate units was identified by 31 P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified
using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed
by visualization of polyP in the dense granules using 4â²,6-diamidino-2-phenylindole and by its release together with pyrophosphate
and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium
and potassium and both bafilomycin A 1 -sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the
fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca 2+ concentration (Ca 2+ i ), they were shown to possess a significant amount of Ca 2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in Ca 2+ i induced by nigericin, monensin, or the weak base, NH 4 Cl, in the nominal absence of extracellular Ca 2 and 2) the effect of ionomycin, which could not take Ca 2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin,
monensin, or NH 4 Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both
by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria
and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to
humans.
We have used isothermal titration calorimetry (ITC) to study the thermodynamics of binding of 12 bisphosphonates to human bone. The ITC results show that there are two binding sites. Site A is the ...weak, highly populated site seen by NMR and is characterized by an average ΔG of binding of −5.2 kcal. Site B is a strong binding site characterized by a ΔG of binding of −8.5 kcal. Binding to both sites is overwhelmingly entropy driven. Using a thermodynamic group approach and a linear regression method, we predict the ΔG of binding of all 12 compounds with an R 2 = 0.95 (a 0.19 kcal error variance estimate, ∼3% of the total ΔG range), opening up the way to designing novel chemotherapy, immunotherapy, and anti-infectious disease drugs having weak bone binding affinity.
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