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SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca2+ homeostasis. Here, we synthesized 19 analogs of SQ109 and tested them against bacteria: M. smegmatis, M. tuberculosis, M. abscessus, Bacillus subtilis and Escherichia coli, as well as against the protozoan parasites, Trypanosoma brucei, T. cruzi, Leishmania donovani, L. mexicana and Plasmodium falciparum. Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct, but two analogs were ~4-8 fold more active than was SQ109 against M. abscessus, including a highly drug resistant strain harboring a A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl and benzyl analogs had 4-10x increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new anti-malarial drug leads. We also used surface plasmon resonance to investigate the binding of inhibitors to MmpL3, and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target, in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, expected to lead to enhanced uncoupler activity.

Terpenes and isoprenoids are the most diverse small organic molecules on Earth. Here, we obtained the structures of sesquisabinene and santalene synthases from Santalum album L. in the presence or ...

Cis-prenyltransferases such as undecaprenyl diphosphate synthase (UPPS) and decaprenyl diphosphate synthase (DPPS) are essential enzymes in bacteria and are involved in cell wall biosynthesis. UPPS and DPPS are absent in the human genome, so they are of interest as targets for antibiotic development. Here, we screened a library of 750 compounds from National Cancer Institute Diversity Set V for the inhibition of Mycobacterium tuberculosis DPPS and found 17 hits, and then IC50s were determined using dose-response curves. Compounds were tested for growth inhibition against a panel of bacteria, for in vivo activity in a Staphylococcus aureus/Caenorhabditis elegans model, and for mammalian cell toxicity. The most active DPPS inhibitor was the dicarboxylic acid redoxal (compound 10), which also inhibited undecaprenyl diphosphate synthase (UPPS) as well as farnesyl diphosphate synthase. 10 was active against S. aureus, Clostridiodes difficile, Bacillus anthracis Sterne, and Bacillus subtilis, and there was a 3.4-fold increase in IC50 on addition of a rescue agent, undecaprenyl monophosphate. We found that 10 was also a weak protonophore uncoupler, leading to the idea that it targets both isoprenoid biosynthesis and the proton motive force. In an S. aureus/C. elegans in vivo model, 10 reduced the S. aureus burden 3 times more effectively than did ampicillin.

Human farnesyl pyrophosphate synthase (Homo sapiens FPPS, HsFPPS) is a target for treating bone resorption diseases and some cancers. HsFPPS is potently inhibited by bisphosphonates, but due to poor cell penetration and distribution in soft tissue, there is currently interest in the development of non-bisphosphonate inhibitors as cancer therapeutics. Here, we report the discovery and development of HsFPPS inhibitors based on the phenolic diterpene carnosic acid (CA), an antimicrobial found in rosemary and sage, which showed better cellular anticancer activities than the bisphosphonate drug zoledronate in pancreatic cancer cell lines, as well as an HsFPPS-dependent mechanism of action. Hit-to-lead optimization of CA improved HsFPPS inhibition by >100-fold. A slow dissociation inhibition pattern and a noncompetitive allosteric binding mode were found, and cellular mechanism-of-action studies showed that these inhibitors inhibit tumor cell growth primarily by inhibiting HsFPPS, leading to downregulation of Ras prenylation and cell apoptosis. The discovery of this series of compounds together with proof-of-mechanism in pancreatic cancer cells may pave the way for targeting HsFPPS in soft tissue cancers using natural-product-derived inhibitors.

SQ109 is a drug candidate for the treatment of tuberculosis (TB). It is thought to target primarily the protein MmpL3 in Mycobacterium tuberculosis, but it also inhibits the growth of some other bacteria, as well as fungi and protozoa. SQ109 is metabolized by the liver, and it has been proposed that some of its metabolites might be responsible for its activity against TB. Here, we synthesized six potential P450 metabolites of SQ109 and used these as well as 10 other likely metabolites as standards in a mass spectrometry study of M. tuberculosis-infected rabbits treated with SQ109, in addition to testing all 16 putative metabolites for anti-bacterial activity. We found that there were just two major metabolites in lung tissue: a hydroxy-adamantyl analog of SQ109 and N’-adamantylethylenediamine. Neither of these, or the other potential metabolites tested, inhibited the growth of M. tuberculosis, or of M. smegmatis, Bacillus subtilis or E. coli, making it unlikely that an SQ109 metabolite contributes to its anti-bacterial activity. In the rabbit TB model, it is thus the gradual accumulation of non-metabolized SQ109 in tissues to therapeutic levels that leads to good efficacy. Our results also provide new insights into how SQ109 binds to its target MmpL3, based on our mass spectroscopy results which indicate that the charge in SQ109 is primarily localized on the geranyl nitrogen, explaining the very short distance to a key Asp found in the X-ray structure of SQ109 bound to MmpL3. Our results also suggest that it is intact SQ109 that is likely to target some of the other bacteria, fungi and protozoa in which MmpL3-like proteins have recently been reported.

The tuberculosis drug candidate SQ109 targets the trehalose monomycolate transporter MmpL3 in Mycobacterium tuberculosis and also has activity against other pathogens. We found related proteins in 22 protozoa, including Trypanosoma cruzi and Entamoeba histolytica, as well as in archaea and other bacteria, including the fatty acid transporter, FarE. We show these proteins, alpha-MMPL proteins, adopt similar structures to that of MsMmpL3 having two sets (P1 and P2) of conserved active site/H+-transporter Asp, Tyr and Phe residues in “pentad” motifs (DYxxF) that can bind to the SQ109 ethylenediamine and adamantyl moieties. Based on structural comparisons with MsMmpL3, we find that there are superimposable transmembrane and H+-transporter structures in much larger proteins, beta-MMPLs, found in apicomplexan parasites, fungi, plants and animals. They also contain double “pentad” motifs in which the P1 Asp is totally conserved, but the P2 Asp may also be a Glu, and the P2 Phe seen in the alpha-MMPLs is a His that H-bonds to the P1 and P2 Asp/Glu residues. There are also 5 conserved Ser/Thr residues that extend the H-bond/H+-transporter network with 2 interacting directly with the P1 Asp, and the His. We propose that all MMPL proteins are involved in proton motive force-mediated lipid (phospholipid, glycolipid, sterol, fatty acid) transport, and that SQ109 may target some pathogens directly, by binding to the P1/P2 motifs. Overall, the results are of general interest since they indicate that there are two major classes of lipid transporters: alpha-MMPL proteins found in many bacteria and protozoa, and much larger, beta-MMPL proteins, found in fungi, apicomplexa, plants and animals and, in some cases, they are potential drug targets.

The tuberculosis drug candidate SQ109 targets the trehalose monomycolate transporter MmpL3 in Mycobacterium tuberculosis and also has activity against other pathogens. We found related proteins in 22 protozoa, including Trypanosoma cruzi and Entamoeba histolytica, as well as in archaea and other bacteria, including the fatty acid transporter, FarE. We show these proteins, alpha-MMPL proteins, adopt similar structures to that of MsMmpL3 having two sets (P1 and P2) of conserved active site/H+-transporter Asp, Tyr and Phe residues in “pentad” motifs (DYxxF) that can bind to the SQ109 ethylenediamine and adamantyl moieties. Based on structural comparisons with MsMmpL3, we find that there are superimposable transmembrane and H+-transporter structures in much larger proteins, beta-MMPLs, found in apicomplexan parasites, fungi, plants and animals. They also contain double “pentad” motifs in which the P1 Asp is totally conserved, but the P2 Asp may also be a Glu, and the P2 Phe seen in the alpha-MMPLs is a His that H-bonds to the P1 and P2 Asp/Glu residues. There are also 5 conserved Ser/Thr residues that extend the H-bond/H+-transporter network with 2 interacting directly with the P1 Asp, and the His. We propose that all MMPL proteins are involved in proton motive force-mediated lipid (phospholipid, glycolipid, sterol, fatty acid) transport, and that SQ109 may target some pathogens directly, by binding to the P1/P2 motifs. Overall, the results are of general interest since they indicate that there are two major classes of lipid transporters: alpha-MMPL proteins found in many bacteria and protozoa, and much larger, beta-MMPL proteins, found in fungi, apicomplexa, plants and animals and, in some cases, they are potential drug targets.

We report that alkyl-subsituted bisphosphonates have activity against Bacillus anthracis Sterne (0.40 µg/mL), Mycobacterium smegmatis (1.4 µg/mL), Bacillus subtilis (1.0 µg/mL) and Staphylococcus aureus (13 µg/mL). In many cases, there is no effect of serum binding, as well as low activity against a human embryonic kidney cell line. Targeting of isoprenoid biosynthesis is involved with74 having IC(50) values of ~100 nM against heptaprenyl diphosphate synthase and 200 nM against a farnesyl diphosphate synthase. Bacillus subtilis growth inhibition was rescued by addition of farnesyl diphosphate, menaquinone-4 (MK-4) or undecaprenyl diphosphate (UP) and the combination of MK-4 plus UP resulted in a 25x increase in ED(50), indicating targeting of both quinone and cell wall biosynthesis. Clostridioides difficile was inhibited by 74 and since this organism does not synthesize quinones, cell wall biosynthesis is the likely target. We also solved three X-ray structures of inhibitors bound to octaprenyl diphosphate and/or undecaprenyl diphosphate synthases.

We report the structures of the terpene cyclases Santalum album L. sesquisabinene synthases 1 and 2 and of santalene synthase, in apo forms, and with the sesquisabinene synthases, bound to either farnesyl diphosphate (FPP), farnesyl S-thiolo-diphosphate, FPP containing a POP bridging O-to-CCl2 substitution, or to sabinene, leading to a sequential mechanism for substrate binding and catalysis. We trapped early pre-catalytic inactive open forms that show how ligands initially bind to the apo-proteins, then when the pocket closes, catalysis can proceed. We also show that there are strong structural similarities between the most highly conserved residues in class I cyclases and those in head-to-tail (aHT) trans-prenyl transferases—outside the well-known DDXXD-like and NSE/DTE-like domains. In the aHT prenyltransferases there is a highly conserved Thr>Gln>Asp>Tyr motif and in the cyclases, a similar Thr>Arg>Asp>Tyr domain, these residues forming very similar, extended H-bond networks (rmsd ~1.4 Å) that are involved in catalysis, leading to the proposal that there are 3 key domains in both the cyclases and the aHT prenyltransferases: The AC-domain that binds MgA and MgC; the B domain that binds MgB and leads to pocket closure, ionization, and condensation or cyclization; and the D-domain H-bond network, involved in H+ elimination. In aHH prenyltransferases the overall folds and MgABC motifs are similar to those found in the cyclase and aHT proteins, but the full Thr>Arg/Gln>Asp>Tyr domain is absent and instead there are Tyr/Asp or Tyr/Glu residues that bind to MgC and are highly conserved. Overall, the results are of general interest since they show unexpected similarities between the enzymes that produce the most diverse molecules on Earth: aHT and aHH prenyltransferases, and terpenoid cyclases.

We report the results of an investigation into the catalytic role of highly conserved amide (asparagine, glutamine) and OH-containing (serine, tyrosine) residues in several prenyltransferases. We first obtained the X-ray structure of cyclolavandulyl diphosphate synthase containing two molecules of the substrate analog dimethylallyl (S)-thiolodiphosphate (DMASPP). The two molecules have similar diphosphate group orientations to those seen in other ζ-fold (cis- head-to-tail and head-to-middle) prenyltransferases with one diphosphate moiety forming a bidentate chelate with Mg(2+) in the so-called S1 site (which is typically the allylic binding site in ζ-fold proteins) while the second diphosphate binds to Mg(2+) in the so-called S2 site (which is typically the homoallylic binding site in ζ-fold proteins) via a single P1O1 oxygen. The latter interaction can facilitate direct phosphate-mediated proton abstraction via P1O2, or more likely by an indirect mechanism in which P1O2 stabilizes a basic asparagine species that removes H(+), which is then eliminated via an Asn-Ser shuttle. The universal occurrence of Asn-Ser pairs in ζ-fold proteins leads to the idea that the highly conserved amide (Asn, Gln) and OH-containing (Tyr) residues seen in many “head-to-head” prenyltransferases such as squalene and dehydrosqualene synthase might play similar roles, in H(+) elimination. Structural, bioinformatics and mutagenesis investigations indeed indicate an important role of these residues in catalysis, with the results of density functional theory calculations showing that Asn bound to Mg(2+) can act as a general (imine-like) base, while Gln, Tyr and H(2)O form a proton channel that is adjacent to the conventional (Asp-rich) “active site”. Taken together, our results lead to mechanisms of proton-elimination from carbocations in numerous prenyltransferases in which neutral species (Asn, Gln, Ser, Tyr, H(2)O) act as proton shuttles, complementing the more familiar roles of acidic groups (in Asp and Glu) that bind to Mg(2+), and basic groups (primarily Arg) that bind to diphosphates, in isoprenoid biosynthesis.

Lysyl oxidase (LOX) is a copper amine oxidase that cross-links collagens and elastin in connective tissue and plays an important role in fibrosis, cancer development, and formation of the "metastatic niche". Despite its important biological functions, the structure of human LOX remains unknown (unlike that of an unrelated LOX, from Pichia pastoris). Here, we expressed active LOX from Drosophila melanogaster, DmLOXL1, a close homologue of human LOX, and characterized it by MS, UV-vis, activity, and inhibition assays. We then used bioinformatics, electron paramagnetic resonance, electron spin-echo envelope modulation, and hyperfine sublevel-correlation (HYSCORE) spectroscopies to probe Cu-ligand bonding finding direct evidence for pH-dependent Cu-His interactions. At pH = 9.3, the spectroscopic data indicated primarily a single His bound to Cu, but at pH = 7.5, there was evidence for a ∼ 1:1 mixture of species containing 1 and 3 His ligands. We then used HYSCORE to probe possible interactions between the LOX inhibitor BAPN (β-aminopropionitrile; 1-[

We synthesized a series of polyoxometalate-bisphosphonate complexes containing MoVIO6 octahedra, zoledronate or an N-alkyl (n-C6 or n-C8) zoledronate analog, and in two cases, Mn as a heterometal. Mo6L2 (L = Zol, ZolC6, ZolC8) and Mo4L2Mn (L = Zol, ZolC8) were characterized by using single-crystal X-ray crystallography and/or IR spectroscopy, elemental and EDX analysis and 31P NMR. We found promising activity against human non-small cell lung cancer (NCI-H460) cells with IC50 values for growth inhibition of ~5 μM per bisphosphonate ligand. The effects of bisphosphonate complexation on IC50 decreased with increasing bisphosphonate chain length: C0 ~6.1x; C6 ~3.4× and C8 ~1.1x. We then determined the activity of one of the most potent compounds in the series, Mo4Zol2Mn(III), against SK-ES-1 sarcoma cells in a mouse xenograft system finding a ~5× decrease in tumor volume than found with the parent compound zoledronate at the same compound dosing (5 μg/mouse). Overall, the results are of interest since we show for the first time that heteropolyoxomolybdate-bisphosphonate hybrids kill tumor cells in vitro and significantly decrease tumor growth, in vivo, opening up new possibilities for targeting both Ras as well as EGFR (epidermal growth factor receptor) driven cancers.

We synthesized a library of 48 analogs of the Mycobacterium tuberculosis cell growth inhibitor SQ109 in which the ethylene diamine linker was replaced by oxa-, thia- or heterocyclic species, and in some cases, the adamantyl group was replaced by a 1,2-carborane or the N-geranyl group by another hydrophobic species. Compounds were tested against Mycobacterium tuberculosis (H37Rv and/or Erdman), Mycobacterium smegmatis, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Trypanosoma brucei and two human cell lines (human embryonic kidney, HEK293T, and the hepatocellular carcinoma, HepG2). Most potent activity was found against T. brucei, the causative agent of human African trypanosomiasis, and involved targeting of the mitochondrial membrane potential with 15 SQ109 analogs being more active than was SQ109 in cell growth inhibition, having IC50 values as low as 12 nM (5.5 ng/mL) and a selectivity index of ~300.

Farnesyl diphosphate synthase (FPPS) is an important drug target for bone resorption, cancer, and some infectious diseases. Here, we report five new structures including two having unique bound ligand geometries. The diamidine inhibitor 7 binds to human FPPS close to the homoallylic (S2) and allosteric (S3) sites and extends into a new site, here called S4. With the bisphosphonate inhibitor 8, two molecules bind to Trypanosoma brucei FPPS, one molecule in the allylic site (S1) and the other close to S2, the first observation of two bisphosphonate molecules bound to FPPS. We also report the structures of apo-FPPS from T. brucei, together with two more bisphosphonate-bound structures (2,9), for purposes of comparison. The diamidine structure is of particular interest because 7 could represent a new lead for lipophilic FPPS inhibitors, while 8 has low micromolar activity against T. brucei, the causative agent of human African trypanosomiasis.

We report the results of an investigation of the activity of a series of amidine and bisamidine compounds against Staphylococcus aureus and Escherichia coli. The most active compounds bound to an AT-rich DNA dodecamer (CGCGAATTCGCG)2 and using DSC were found to increase the melting transition by up to 24 °C. Several compounds also inhibited undecaprenyl diphosphate synthase (UPPS) with IC50 values of 100-500 nM, and we found good correlations (R(2) = 0.89, S. aureus; R(2) = 0.79, E. coli) between experimental and predicted cell growth inhibition by using DNA ΔTm and UPPS IC50 experimental results together with one computed descriptor. We also solved the structures of three bisamidines binding to DNA as well as three UPPS structures. Overall, the results are of general interest in the context of the development of resistance-resistant antibiotics that involve multitargeting.

(E)-1-Hydroxy-2-methylbut-2-enyl 4-diphosphate reductase (IspH) is a [Fe4S4] cluster-containing enzyme involved in isoprenoid biosynthesis in many bacteria as well as in malaria parasites and is an important drug target. Several inhibitors including amino and thiol substrate analogues, as well as acetylene and pyridine diphosphates, have been reported. Here, we investigate the mode of binding of four pyridine diphosphates to Escherichia coli IspH by using X-ray crystallography. In three cases, one of the iron atoms in the cluster is absent, but in the structure with (pyridin-3-yl)methyl diphosphate, the most potent pyridine-analogue inhibitor reported previously, the fourth iron of the [Fe4S4] cluster is present and interacts with the pyridine ring of the ligand. Based on the results of quantum chemical calculations together with the crystallographic results we propose a side-on η(2) coordination of the nitrogen and the carbon in the 2-position of the pyridine ring to the unique fourth iron in the cluster, which is in the reduced state. The X-ray structure enables excellent predictions using density functional theory of the (14)N hyperfine coupling and quadrupole coupling constants reported previously using HYSCORE spectroscopy, as well as providing a further example of the ability of such [Fe4S4]-containing proteins to form organometallic complexes.

The [4Fe-4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and (2)H, (17)O, and (57)Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin, thioredoxin reductase, in that one electron is transferred to the [4Fe-4S](2+) cluster, which then performs a formal two-electron reduction of its substrate, generating an oxidized high potential iron-sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate's 3-CH(2)OH group has rotated away from the reduced iron-sulfur cluster, and the next, η(3)-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogues (4 and 5) follow the same reaction mechanism.

We report the synthesis and characterization of eight new Mo, W, or V-containing polyoxometalate (POM) bisphosphonate complexes with metal nuclearities ranging from 1 to 6. The compounds were synthesized in water by treating Mo(VI), W(VI), V(IV), or V(V) precursors with biologically active bisphosphonates H(2)O(3)PC(R)(OH)PO(3)H(2) (R = C(3)H(6)NH(2), Ale; R = CH(2)S(CH(3))(2), Sul and R = C(4)H(5)N(2), Zol, where Ale = alendronate, Sul = (2-Hydroxy-2,2-bis-phosphono-ethyl)-dimethyl-sulfonium and Zol = zoledronate). Mo(6)(Sul)(2) and Mo(6)(Zol)(2) contain two trinuclear Mo(VI) cores which can rotate around a central oxo group while Mo(Ale)(2) and W(Ale)(2) are mononuclear species. In V(5)(Ale)(2) and V(5)(Zol)(2) a central V(IV) ion is surrounded by two V(V) dimers bound to bisphosphonate ligands. V(6)(Ale)(4) can be viewed as the condensation of one V(5)(Ale)(2) with one additional V(IV) ion and two Ale ligands, while V(3)(Zol)(3) is a triangular V(IV) POM. These new POM bisphosphonates complexes were all characterized by single-crystal X-ray diffraction. The stability of the Mo and W POMs was studied by (31)P NMR spectroscopy and showed that all compounds except the mononuclear Mo(Ale)(2) and W(Ale)(2) were stable in solution. EPR measurements performed on the vanadium derivatives confirmed the oxidation state of the V ions and evidenced their stability in aqueous solution. Electrochemical studies on V(5)(Ale)(2) and V(5)(Zol)(2) showed reduction of V(V) to V(IV), and magnetic susceptibility investigations on V(3)(Zol)(3) enabled a detailed analysis of the magnetic interactions. The presence of zoledronate or vanadium correlated with the most potent activity (IC(50)~1-5 μM) against three human tumor cell lines.

We report the discovery of antibacterial leads, keto- and diketo-acids, targeting two prenyl transferases: undecaprenyl diphosphate synthase (UPPS) and dehydrosqualene synthase (CrtM). The leads were suggested by the observation that keto- and diketo-acids bind to the active site Mg2+/Asp domain in HIV-1 integrase, and similar domains are present in prenyl transferases. We report the X-ray crystallographic structures of one diketo-acid and one keto-acid bound to CrtM, which supports the Mg2+ binding hypothesis, together with the X-ray structure of one diketo-acid bound to UPPS. In all cases, the inhibitors bind to a farnesyl diphosphate substrate-binding site. Compound 45 had cell growth inhibition MIC90 values of ∼250–500 ng/mL against Staphylococcus aureus, 500 ng/mL against Bacillus anthracis, 4 μg/mL against Listeria monocytogenes and Enterococcus faecium, and 1 μg/mL against Streptococcus pyogenes M1 but very little activity against Escherichia coli (DH5α, K12) or human cell lines.

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 drugs--such as Lipitor (used to lower cholesterol), Fosamax (used to treat osteoporosis), and many anti-infectives--target 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" (η(2)-alkenes) and η(1)/η(3)-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 clearance--a 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.

We report the inhibition of the Aquifex aeolicus IspH enzyme (LytB, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, EC 1.17.1.2) by a series of diphosphates and bisphosphonates. The most active species was an alkynyl diphosphate having IC(50) = 0.45 microM (K(i) approximately 60 nM), which generated a very large change in the 9 GHz EPR spectrum of the reduced protein. On the basis of previous work on organometallic complexes, together with computational docking and quantum chemical calculations, we propose a model for alkyne inhibition involving pi (or pi/sigma) "metallacycle" complex formation with the unique fourth Fe in the Fe(4)S(4) cluster. Aromatic species had less activity, and for these we propose an inhibition model based on an electrostatic interaction with the active site E126. Overall, the results are of broad general interest since they not only represent the first potent IspH inhibitors but also suggest a conceptually new approach to inhibiting other Fe(4)S(4)-cluster-containing proteins that are of interest as drug and herbicide targets.

The gold color of Staphylococcus aureus is derived from the carotenoid staphyloxanthin, a virulence factor for the organism. Here, we report the synthesis and activity of a broad variety of staphyloxanthin biosynthesis inhibitors that inhibit the first committed step in its biosynthesis, condensation of two farnesyl diphosphate (FPP) molecules to dehydrosqualene, catalyzed by the enzyme dehydrosqualene synthase (CrtM). The most active compounds are phosphonoacetamides that have low nanomolar K(i) values for CrtM inhibition and are active in whole bacterial cells and in mice, where they inhibit S. aureus disease progression. We also report the X-ray crystallographic structure of the most active compound, N-3-(3-phenoxyphenyl)propylphosphonoacetamide (IC(50) = 8 nM, in cells), bound to CrtM. The structure exhibits a complex network of hydrogen bonds between the polar headgroup and the protein, while the 3-phenoxyphenyl side chain is located in a hydrophobic pocket previously reported to bind farnesyl thiodiphosphate (FsPP), as well as biphenyl phosphonosulfonate inhibitors. Given the good enzymatic, whole cell, and in vivo pharmacologic activities, these results should help guide the further development of novel antivirulence factor-based therapies for S. aureus infections.

We report the X-ray structures of several bisphosphonate inhibitors of geranylgeranyl diphosphate synthase, a target for anticancer drugs. Bisphosphonates containing unbranched side chains bind to either the farnesyl diphosphate (FPP) substrate site, the geranylgeranyl diphosphate (GGPP) product site, and in one case, both sites, with the bisphosphonate moiety interacting with 3 Mg (2+) that occupy the same position as found in FPP synthase. However, each of three "V-shaped" bisphosphonates bind to both the FPP and GGPP sites. Using the Glide program, we reproduced the binding modes of 10 bisphosphonates with an rms error of 1.3 A. Activities of the bisphosphonates in GGPPS inhibition were predicted with an overall error of 2x by using a comparative molecular similarity analysis based on a docked-structure alignment. These results show that some GGPPS inhibitors can occupy both substrate and product site and that binding modes as well as activity can be accurately predicted, facilitating the further development of GGPPS inhibitors as anticancer agents.

We report the results of an investigation of the binding of a series of bisphosphonate drugs to human bone using 2H, 13C, 15N, and 31P nuclear magnetic resonance spectroscopy. The 31P NMR results show that the bisphosphonate groups bind irrotationally to bone, displacing orthophosphate from the bone mineral matrix. Binding of pamidronate is well described by a Langmuir-like isotherm, from which we deduce an approximately 30-38 A2 surface area per pamidronate molecule and a deltaG = -4.3 kcal mol(-1). TEDOR of [13C3, 15N] pamidronate on bone shows that the bisphosphonate binds in a gauche [N-C(1)] conformation. The results of 31P as well as 15N shift and cross-polarization measurements indicate that risedronate binds weakly, since it has a primarily neutral pyridine side chain, whereas zoledronate (with an imidazole ring) binds more strongly, since the ring is partially protonated. The results of 2H NMR measurements of side-chain 2H-labeled pamidronate, alendronate, zoledronate, and risedronate on bone show that all side chains undergo fast but restricted motions. In pamidronate, the motion is well simulated by a gauche+/gauche- hopping motion of the terminal -CH2-NH3(+) group, due to jumps from one anionic surface group to another. The results of double-cross polarization experiments indicate that the NH3(+)-terminus of pamidronate is close to the bone mineral surface, and a detailed model is proposed in which the gauche side-chain hops between two bone PO4(3-) sites.

We investigated three series of sulfonium bisphosphonates for their activity in inhibiting the growth of three human tumor cell lines. The first series consisted of 6 cyclic sulfonium bisphosphonates, the most active species having an (average) IC50 of 89 microM. The second consisted of 10 phenylalkyl and phenylalkoxy bisphosphonates, the most active species having an IC50 of 18 microM. The third series consisted of 17 n-alkyl sulfonium bisphosphonates, the most active species having an IC50 of approximately 240 nM. Three QSAR models showed that the experimental cell growth inhibition results could be well predicted. We also determined the structures of one sulfonium bisphosphonate bound to farnesyl diphosphate synthase, finding that it binds exclusively to the dimethylallyl diphosphate binding site. These results are of interest since they show that sulfonium bisphosphonates can have potent activity against a variety of tumor cell lines, the most active species having IC50 values much lower than conventional nitrogen-containing bisphosphonates.

We report the results of a solid-state NMR and quantum chemical investigation of the 13C gamma NMR chemical shifts in phenylalanine and tyrosine in dipeptides and proteins. Accurate computation of the experimental shifts is shown to require a good description of local electrostatic field effects, and we find the best results (R2=0.94, rmsd=1.6 ppm, range = 17.1 ppm, N=14) by using a self-consistent reaction field continuum model. There are no obvious correlations with phi, psi, chi 1, or chi2 torsion angles, unlike the results seen with other amino acids. There is, however, a linear relation between computed C gamma atomic charges and shifts for the 14 peptide as well as 18 protein residues investigated. This result is similar to the correlation reported in the 1960s between pi-electron density and 13C shifts for classical 4n + 2 (n=0, 1, 2) pi-electron aromatic species, such as cyclopentadienide and the tropylium cation, and in fact, we found that the shielding/atomic charge correlation seen in the peptides and proteins is virtually identical to that seen with a broad range of aromatic carbocations/carbanions. These results suggest the dominance of an electrostatic field polarization model in which increasing pi electron density results in an increase in C gamma atomic charge and increased shielding (of sigma 11 and sigma 22, perpendicular to the pi orbital) in Phe and Tyr, as well as in the other aromatic species. These results are of general interest since they demonstrate the importance of electrostatic field effects on Phe and Tyr C gamma chemical shifts in peptides and proteins and imply that inclusion of these effects will be necessary in order to interpret the shifts of other aromatic species, such as drug molecules, bound to proteins.

We have studied the effects of CO coverage on surface diffusion rates of CO adsorbed on nanoparticle Pt catalysts in sulfuric acid media by using 13C electrochemical nuclear magnetic resonance spectroscopy (EC-NMR) in the temperature range 253 - 293 K. For CO coverage from θ = 1.0 to 0.36, the diffusion coefficients follow Arrhenius behavior and both activation energy (Ed) and pre-exponential factor (Dco) show CO coverage dependence. Ed increases from 6.0 to 8.4 kcal/mol and DCO varies from 1.1 X 10-8 to 3.7 X 10-6 cm2/s when the coverage is increased from θ = 0.36 to θ = 1.0. On the Pt catalyst surface at partial CO coverage, our data strongly support the free site hopping model of adsorbed CO as the major surface diffusion mechanism, unlike the situation found with a fully CO covered surface where CO exchange between different surface sites is believed to be the major diffusion mechanism. Our results also indicate that the contributions of lateral repulsive interactions exert a stronger influence on the diffusive motion than does the nature of the surface structure. When the diffusion coefficient was estimated from CO stripping measurements by using an electrochemical modeling protocol, the estimated diffusion coefficients were a few orders of magnitude larger than those obtained from the EC-NMR experiments. Overall these results are important for improving our understanding of electrochemical surface dynamics of molecules at interfaces, and may help facilitate better control of fuel cell reactions where the presence of surface CO plays a crucial role in controlling the reaction rates.

Bisphosphonates are a class of molecules in widespread use in treating bone resorption diseases and are also of interest as immunomodulators and anti-infectives. They function by inhibiting the enzyme farnesyl diphosphate synthase (FPPS), but the details of how these molecules bind are not fully understood. Here, we report the results of a solid-state (13)C, (15)N, and (31)P magic-angle sample spinning (MAS) NMR and quantum chemical investigation of several bisphosphonates, both as pure compounds and when bound to FPPS, to provide information about side-chain and phosphonate backbone protonation states when bound to the enzyme. We then used computational docking methods (with the charges assigned by NMR) to predict how several bisphosphonates bind to FPPS. Finally, we used X-ray crystallography to determine the structures of two potent bisphosphonate inhibitors, finding good agreement with the computational results, opening up the possibility of using the combination of NMR, quantum chemistry and molecular docking to facilitate the design of other, novel prenytransferase inhibitors.

A general protocol for the structural characterization of paramagnetic molecular solids using solid-state NMR is provided and illustrated by the characterization of a high-spin Fe(II) catalyst precursor. We show how good NMR performance can be obtained on a molecular powder sample at natural abundance by using very fast (30 kHz) magic angle spinning (MAS), even though the individual NMR resonances have highly anisotropic shifts and very short relaxation times. The results include the optimization of broadband heteronuclear (proton-carbon) recoupling sequences for polarization transfer; the observation of single or multiple quantum correlation spectra between coupled spins as a tool for removing the inhomogeneous bulk magnetic susceptibility (BMS) broadening; and the combination of NMR experiments and density functional theory calculations, to yield assignments.

We have investigated M···H−C interactions in d8 square planar Rh and Pt complexes involving different types of C−H bonds, as well as a M···H−N hydrogen-bonded complex, using a combination of DFT and “atoms-in-molecules” (AIM) theory. The AIM analysis shows that both M···H−C and M···H−N interactions in d8 square planar complexes are of a closed-shell, electrostatic nature, similar to protein backbone hydrogen bonds. However, at the shortest M···H distances, both interactions have partial covalence. We find no evidence for the involvement of dz2 orbitals in M···H−C interactions but do find evidence for the involvement of dxz/yz orbitals in M···H−C bonding. The DFT calculations reproduce well the experimental proton NMR chemical shifts, with a theory-versus-experiment correlation coefficient R2 = 0.985. There are large downfield 1H NMR chemical shift changes on metal bonding, accompanied by changes in shielding tensor orientations.

Trypanosoma brucei, the causative agent of African trypanosomiasis, contains a soluble, vacuolar pyrophosphatase, TbVSP1, not present in humans, which is essential for the growth of bloodstream forms in their mammalian host. Here, we report the inhibition of a recombinant TbVSP1 expressed in Escherichia coli by a panel of 81 bisphosphonates. The IC50 values were found to vary from approximately 2 to 850 microM. We then used 3D QSAR (comparative molecular field and comparative molecular similarity index; CoMFA and CoMSIA) methods to analyze the enzyme inhibition results. The R2 values for the experimental versus the QSAR-predicted activities were 0.78 or 0.61 for CoMFA and 0.79 or 0.68 for CoMSIA, for two different alignments. The root-mean-square (rms) pIC50 error for the best CoMFA model was 0.41 for five test sets of five activity predictions, which translates to a factor of approximately 2.6 error in IC50 prediction. For CoMSIA, the rms pIC50 error and error factors were 0.35 and 2.2, respectively. In general, the most active compounds contained both a single aromatic ring and a hydrogen bond donor feature. Thirteen of the more potent compounds were then tested in vivo in a mouse model of T. brucei infection. The most active compound in vivo provided a 40% protection from death with no apparent side effects, suggesting that further development of such compounds may be of interest.

We report the first solid-state NMR, crystallographic, and quantum chemical investigation of the origins of the 13C NMR chemical shifts of the imidazole group in histidine-containing dipeptides. The chemical shift ranges for Cgamma and Cdelta2 seen in eight crystalline dipeptides were very large (12.7-13.8 ppm); the shifts were highly correlated (R2= 0.90) and were dominated by ring tautomer effects and intermolecular interactions. A similar correlation was found in proteins, but only for buried residues. The imidazole 13C NMR chemical shifts were predicted with an overall rms error of 1.6-1.9 ppm over a 26 ppm range, by using quantum chemical methods. Incorporation of hydrogen bond partner molecules was found to be essential in order to reproduce the chemical shifts seen experimentally. Using AIM (atoms in molecules) theory we found that essentially all interactions were of a closed shell nature and the hydrogen bond critical point properties were highly correlated with the N...H...O (average R2= 0.93) and Nepsilon2...H...N (average R2= 0.98) hydrogen bond lengths. For Cepsilon1, the 13C chemical shifts were also highly correlated with each of these properties (at the Nepsilon2 site), indicating the dominance of intermolecular interactions for Cepsilon1. These results open up the way to analyzing 13C NMR chemical shifts, tautomer states (from Cdelta2, Cepsilon1 shifts), and hydrogen bond properties (from Cepsilon1 shifts) of histidine residue in proteins and should be applicable to imidazole-containing drug molecules bound to proteins, as well.

We have investigated the activity of 60 bisphosphonates against the replication of Toxoplasma gondii in vitro and of three of the most active compounds, in vivo. The two most active compounds found were n-alkyl bisphosphonates containing long (n = 9 or 10) hydrocarbon chains, not the nitrogen-containing species used in bone resorption therapy. The target of all of the most active bisphosphonates appears to be the isoprene biosynthesis pathway enzyme farnesyl pyrophosphate synthase (FPPS), as indicated by the correlations between T. gondii growth inhibition and FPPS (human and Leishmania major) enzyme inhibition and by the fact that a T. gondii strain engineered to overexpress FPPS required considerably higher levels of bisphosphonates to achieve 50% growth inhibition, while the IC(50) for atovaquone (which does not inhibit FPPS) remained the same in the overexpressing strain. The phosphonate inhibitor of the non-mevalonate pathway, fosmidomycin, which inhibits the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase, had no effect on T. gondii growth. To investigate structure-activity relationships (SARs) in more detail, we used two three-dimensional quantitative SAR methods: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), to investigate all 60 bisphosphonates. Both the CoMFA and CoMSIA models indicated a 60-70% contribution from steric interactions and a 30-40% contribution from electrostatic interactions and using four N = 55 training sets for each method, we found on average between a factor of 2 and 3 error in IC(50) prediction. The three most active compounds found in vitro were tested in vivo in a Smith-Webster mouse model and the two most active bisphosphonates were found to provide up to an 80% protection from death, a considerable improvement over that found previously with nitrogen-containing bisphosphonates. This effect may originate in the much higher therapeutic indices of these alkyl bisphosphonates, as deduced from in vitro assays using LD(50) values for growth inhibition of a human cell line. Overall, these results indicate that alkyl bisphosphonates are promising compounds for further development as agents against Toxoplasma gondii growth, in vivo.

We report the design, synthesis and testing of a series of novel bisphosphonates, pyridinium-1-yl-hydroxy-bisphosphonates, based on the results of comparative molecular similarity indices analysis and pharmacophore modeling studies of farnesyl diphosphate synthase (FPPS) inhibition, human Vgamma2Vdelta2 T cell activation and bone resorption inhibition. The most potent molecules have high activity against an expressed FPPS from Leishmania major, in Dictyostelium discoideum growth inhibition, in gammadelta T cell activation and in an in vitro bone resorption assay. As such, they represent useful new leads for the discovery of new bone resorption, antiinfective and anticancer drugs.

Spontaneous deposition of Pd onto catalytic grade Pt nanoparticles has been shown to yield Pt/Pd catalysts having enhanced catalytic activity toward formic acid oxidation, when compared to pure Pt- and Pd-black. Here, we report the results of electrochemical nuclear magnetic resonance (EC NMR) and electrochemical measurements of CO chemisorbed onto these Pt/Pd catalysts, to probe the nature of the CO chemisorption bond, as well as the motional behavior of adsorbed CO. The 13C NMR spectra are broad and can be deconvoluted into two peaks, assigned to CO adsorbed on Pt and Pd sites. From the temperature dependence of the spin−lattice relaxation rates, we conclude that CO chemisorbed on Pd undergoes fast diffusion. The activation energy (Ea) obtained from these results for CO on Pd is smaller than that found for CO adsorbed onto Pd nanoparticles supported on alumina. A two-band model analysis of the NMR data shows that the 5σ orbital of CO makes a significant contribution to the chemisorption bond of CO on Pd...

We report the results of a quantum chemical investigation of the 31P nuclear magnetic resonance (NMR) isotropic chemical shieldings (σiso) and the 31P NMR chemical shielding tensor principal values...

We have obtained the carbon-13 nuclear magnetic resonance spectra of a series of tryptophan-containing peptides and model systems, together with their X-ray crystallographic structures, and used quantum chemical methods to predict the (13)C NMR shifts (or shieldings) of all nonprotonated aromatic carbons (C(gamma), C(delta 2) and C(epsilon 2). Overall, there is generally good accord between theory and experiment. The chemical shifts of Trp C(gamma) in several proteins, hen egg white lysozyme, horse myoglobin, horse heart cytochrome c, and four carbonmonoxyhemoglobins, are also well predicted. The overall Trp C(gamma) shift range seen in the peptides and proteins is 11.4 ppm, and individual shifts (or shieldings) are predicted with an rms error of approximately 1.4 ppm (R value = 0.86). Unlike C(alpha) and N(H) chemical shifts, which are primarily a function of the backbone phi,psi torsion angles, the Trp C(gamma) shifts are shown to be correlated with the side-chain torsion angles chi(1) and chi(2) and appear to arise, at least in part, from gamma-gauche interactions with the backbone C' and N(H) atoms. This work helps solve the problem of the chemical shift nonequivalences of nonprotonated aromatic carbons in proteins first identified over 30 years ago and opens up the possibility of using aromatic carbon chemical shift information in structure determination.

We report the activities of 62 bisphosphonates as inhibitors of the Leishmania major mevalonate/isoprene biosynthesis pathway enzyme, farnesyl pyrophosphate synthase. The compounds investigated exhibit activities (IC(50) values) ranging from approximately 100 nM to approximately 80 microM (corresponding to K(i) values as low as 10 nM). The most active compounds were found to be zoledronate (whose single-crystal X-ray structure is reported), pyridinyl-ethane-1-hydroxy-1,1-bisphosphonates or picolyl aminomethylene bisphosphonates. However, N-alicyclic aminomethylene bisphosphonates, such as incadronate (N-cycloheptyl aminomethylene bisphosphonate), as well as aliphatic aminomethylene bisphosphonates containing short (n = 4, 5) alkyl chains, were also active, with IC(50) values in the 200-1700 nM range (corresponding to K(i) values of approximately 20-170 nM). Bisphosphonates containing longer or multiple (N,N-) alkyl substitutions were inactive, as were aromatic species lacking an o- or m-nitrogen atom in the ring, or possessing multiple halogen substitutions or a p-amino group. To put these observations on a more quantitative structural basis, we used three-dimensional quantitative structure-activity relationship techniques: comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA), to investigate which structural features correlated with high activity. Training set results (N = 62 compounds) yielded good correlations with each technique (R(2) = 0.87 and 0.88, respectively), and were further validated by using a training/test set approach. Test set results (N = 24 compounds) indicated that IC(50) values could be predicted within factors of 2.9 and 2.7 for the CoMFA and CoMSIA methods, respectively. The CoMSIA fields indicated that a positive charge in the bisphosphonate side chain and a hydrophobic feature contributed significantly to activity. Overall, these results are of general interest since they represent the first detailed quantitative structure-activity relationship study of the inhibition of an expressed farnesyl pyrophosphate synthase enzyme by bisphosphonate inhibitors and that the activity of these inhibitors can be predicted within about a factor of 3 by using 3D-QSAR techniques.

We have carried out a series of 195Pt and 13C NMR spectroscopic and electrochemical experiments on commercial Pt−Ru alloy nanoparticles and compared the results with those on Pt-black samples having similar particle sizes. The Pt NMR spectrum of the alloy nanoparticles consists of a single Gaussian peak, completely different from the broad “multi-Gaussian” NMR spectra, which are generally observed for carbon-supported Pt catalysts. Spin−echo decay measurements show that the intrinsic spin−spin relaxation time (T2) is much larger in the alloy compared to Pt-black. A “slow-beat” is observed in the spin−echo decay curve of the alloy, implying that the NMR frequencies of spin−spin coupled Pt nuclei in the alloy nanoparticles are quite similar, unlike the situation found with Pt-black. These 195Pt NMR results strongly suggest that there is a surface enrichment of Pt atoms in the Pt−Ru alloy nanoparticles. The CO-stripping cyclic voltammogram (CV) of the Pt−Ru alloy nanoparticles is broader than that observed w...

The 57Fe Mossbauer quadrupole splittings (ΔEQ) and isomer shifts (δFe) in 3-coordinate high-spin Fe(II) complexes are unusually small, and previous attempts to reproduce their ΔEQ values have been unsuccessful. We show here that, by using large structural models and basis sets, both ΔEQ and δFe values can be quite accurately predicted by using density functional theory. Four systems were investigated: the three 3-coordinate species [LFeX]0 (L = β-diketiminate; X = Cl-, CH3-) and [Fe(SC6H2-2,4,6-tBu3)3]-, in addition to an uncommon 2-coordinate high-spin ferrous thiolate, [Fe(SC6H3-2,6-mes2)2] (mes = mesityl = 2,4,6-Me3C6H2). Both Gaussian-type-orbital and Slater-type-orbital basis sets were investigated, and both yielded ΔEQ and δFe values in good accord with experiment. There were no improvements in these property predictions when (approximate) relativistic effects were included in the calculations. An MO analysis provided a detailed picture of the origin of the small ΔEQ values seen in the 3-coordinate...

We have used quantitative structure-activity relationship (QSAR) techniques, together with pharmacophore modeling, to investigate the relationships between the structures of a wide variety of geminal bisphosphonates and their activity in inhibiting osteoclastic bone resorption. For aryl-X (X = alkyl, oxyalkyl, and sulfanylalkyl) derivatives of pamidronate and one alendronate, a molecular field analysis (MFA) yielded an R(2) value of 0.900 and an F-test of 54 for a training set of 29 compounds. Using reduced training sets, the activities of 20 such compounds were predicted with an average error of 2.1 over a 4000x range in activity. Such good results were only obtained when using the X-ray crystallographic structure of farnesyl pyrophosphate (FPP) bound to the target enzyme, farnesyl pyrophosphate synthase (FPP synthase), to guide the initial molecular alignment. For a series of heterocyclic bisphosphonates, use of the MFA method yielded an R(2) of 0.873 and an F-test of 36 for a training set of 26 compounds. Using a reduced training set, the activities of 20 compounds were predicted with an average error of 2.5 over a 2000x range in activity. With the heterocyclic compounds, test calculations indicated the importance of correct choice of protonation of the heterocyclic rings. For example, thiazoles, pyrazoles, and triazoles have low ( approximately 2-3) pK(a) values and the derived bisphosphonates are inactive in bone resorption since they cannot readily be side chain protonated and are thus poor carbocation reactive intermediate analogues. On the other hand, aminothiazoles, imidazoles, pyridyl, and aminopyridyl species typically have pK(a) values in the range approximately 5-9 and, in the absence of unfavorable steric interactions, the corresponding bisphosphonates are generally good inhibitors. However, aminoimidazole bisphosphonates are generally less active, since their pK(a)s ( approximately 11) are so high, due to guanidinium-like resonance, that they cannot readily be deprotonated, which we propose results in poor cellular uptake. The results of pharmacophore modeling using the Catalyst program revealed the importance of two negative ionizable and one positive charge feature for both aryl-X and heterocyclic pharmacophores, together with the presence of a distal hydrophobic feature in the aryl bisphosphonate and a more proximal aromatic feature in the heterocyclic bisphosphonate pharmacophores. When taken together, these results show that it is now possible to predict the activity, within a factor of about 2.3, of a wide range of aryl-X and heterocyclic bisphosphonates. The results emphasize the importance of utilizing crystallographic structural information to guide the initial alignment of extended bisphosphonates, and in the case of heterocyclic bisphosphonates, the importance of side chain protonation state. These simple ideas may facilitate the design of other, novel bisphosphonates, of use in bone resorption therapy, and as antiparasitic and immunotherapeutic agents.

We report the results of density functional theory (DFT) calculations of the 57Fe Mossbauer quadrupole splittings ΔEQ and isomer shifts δFe of both the high-spin (HS) and low-spin (LS) states of tw...

gammadelta T cells help contribute to innate immunity and are activated by the natural phosphoantigens produced by the organisms responsible for causing, for example, tuberculosis, malaria, tularemia, and plague. They are also activated by synthetic phosphoantigens and are cytotoxic to tumor cells. Here, we show that it is now possible to accurately predict gammadelta T cell activation by both natural and synthetic phosphoantigens by using the quantitative structure-activity relationship (QSAR) techniques commonly used in drug design. This approach should be of use in developing novel immunotherapeutic agents as well as contributing to a better understanding of the immune system's response to infectious agents.

It is well-known that platinum/ruthenium fuel cell catalysts show enhanced CO tolerance compared to pure platinum electrodes, but the reasons are still being debated. We have combined cyclic voltammetry (CV), temperature programmed desorption (TPD), electrochemical nuclear magnetic resonance, and radio active labeling to probe the origin of the ruthenium enhancement in Pt electrodes modified through Ru deposition. The results prove that the addition of ruthenium not only modifies the electronic structure of all the platinum atoms but also leads to the creation of a new form of adsorbed CO. This new form of CO may be ascribed to CO chemisorbed onto the “Ru” region of the electrode surface. TPD and CV results show that the binding of hydrogen is substantially modified due to the presence of Ru. Surprisingly though, TPD indicates that the binding energy of CO on platinum is only weakly affected. Therefore, the changes in the bond energy of CO due to the ligand effect only play a small role in enhancing CO to...

We report the results of 3D-QSAR/CoMFA investigations of the activity of bisphosphonate drugs, farnesyl pyrophosphate synthase (FPPSase) inhibitors, in the inhibition of bone resorption as well as the growth of Dictyostelium discoideum. In the case of D. discoideum, we find an experimental versus QSAR predicted pIC50 R2 value of 0.94 for 16 bisphosphonates over the 9−1200 μM range of IC50 values, a cross-validated R2 = 0.90, and a bootstrapped R2 = 0.94, and we demonstrate that this approach has predictive utility (a 0.18 pIC50 rms error for three test sets of 3 predictions). In bone resorption, we find an experimental versus predicted pLED (lowest effective dose) R2 = 0.79 for 35 bisphosphonates over the 0.0001−1 mg of P/kg LED range, a cross-validated R2 = 0.75, and a bootstrapped R2 = 0.79. Two sets of 31 compounds were used as training sets for the predicted pLED values for two sets of 4 compounds which have an rms error of 0.44, larger than that found with D. discoideum. However, this can be attribut...

We report the results of density functional theory (DFT) calculations of the (57)Fe Mössbauer isomer shifts (delta(Fe)) for a series of 24 inorganic, organometallic, and metalloprotein/metalloporphyrin model systems in S = 0, (1)/(2), 1, (3)/(2), 2, and (5)/(2) spin states. We find an excellent correlation between calculation and experiment over the entire 2.34 mm s(-1) range of isomer shifts: a 0.07-0.08 mm s(-1) rms deviation between calculation and experiment (corresponding to 3-4% of the total delta(Fe) range, depending on the functionals used) with R(2) values of 0.973 and 0.981 (p0.0001). The best results are obtained by using the hybrid exchange-correlation functional B3LYP, used previously for (57)Fe Mössbauer quadrupole splittings and (57)Fe NMR chemical shifts and chemical shielding anisotropies. The relativistically corrected value of alpha, alpha(rel), converges with the large basis set used in this work, but the exact values vary somewhat with the methods used: -0.253 a(0)(3) mm s(-1) (Hartree-Fock; HF); -0.316 a(0)(3) mm s(-1) (hybrid HF-DFT; B3LYP), or -0.367 a(0)(3) mm s(-1) (pure DFT; BPW91). Both normal and intermediate spin state isomer shifts are well reproduced by the calculations, as is the broad range of delta(Fe) values: from [Fe(VI)O(4)](2-) (-0.90 mm s(-1) expt; -1.01 mm s(-1) calc) to KFe(II)F(3) (1.44 mm s(-1) expt; 1.46 mm s(-1) calc). Molecular orbital analyses of all inorganic solids as well as all organometallic and metalloporphyrin systems studied reveal that there are three major core MO contributions to rho(tot)(0), the total charge density at the iron nucleus (and hence delta(Fe)), that do not vary with changes in chemistry, while the valence MO contributions are highly correlated with delta(Fe) (R(2) = 0.915-0.938, depending on the functionals used), and the correlation between the valence MO contributions and the total MO contribution is even better (R(2) = 0.965-0.976, depending on the functionals used). These results are of general interest since they demonstrate that DFT methods now enable the accurate prediction of delta(Fe) values in inorganic, organometallic, and metalloporphyrin systems in all spin states and over a very wide range of delta(Fe) values with a very small rms error.

We report the results of a comparative molecular field analysis (CoMFA) investigation of the growth inhibition of the bloodstream form of Trypanosoma brucei rhodesiense trypomastigotes by bisphosphonates. A quantitative three-dimensional structure-activity relationship CoMFA model for a set of 26 bisphosphonates having a range of activity spanning approximately 3 orders of magnitude (minimum IC(50) = 220 nM; maximum IC(50) = 102 microM) yielded an R(2) value of 0.87 with a cross-validated R(2) value of 0.79. The predictive utility of this approach was tested for three sets of three compounds: the average pIC(50) error was 0.23. For the nitrogen-containing bisphosphonates, in general, the activity was aromatic-aliphatic-containing side chains. The activity of aromatic species lacking an alkyl ring substitution decreased from ortho to meta to para substitution; halogen substitutions also reduced activity. For the aliphatic bisphosphonates, the IC(50) values decreased nearly monotonically with increasing chain length (down to IC(50) = 2.0 microM for the n-C(11) alkyl side chain species). We also show, using a "rescue" experiment, that the molecular target of the nitrogen-containing bisphosphonate, risedronate, in T. b. rhodesiense is the enzyme farnesyl pyrophosphate synthase. In addition, we report the LD(50) values of bisphosphonates in a mammalian cell general toxicity screen and present a comparison between the therapeutic indices and the IC(50) values in the T. b. rhodesiense growth inhibition assay. Several bisphosphonates were found to have large therapeutic indices (or =200:1) as well as low IC(50) values, suggesting their further investigation as antiparasitic agents against T. b. rhodesiense.

We report the inhibition of a human recombinant geranylgeranyl diphosphate synthase (GGPPSase) by 23 bisphosphonates and six azaprenyl diphosphates. The IC50 values range from 140 nM to 690 microM. None of the nitrogen-containing bisphosphonates that inhibit farnesyl diphosphate synthase were effective in inhibiting the GGPPSase enzyme. Using three-dimensional quantitative structure-activity relationship/comparative molecular field analysis (CoMFA) methods, we find a good correlation between experimental and predicted activity: R2 = 0.938, R(cv)2 = 0.900, R(bs)2 = 0.938, and F-test = 86.8. To test the predictive utility of the CoMFA approach, we used three training sets of 25 compounds each to generate models to predict three test sets of three compounds. The rms pIC50 error for the nine predictions was 0.39. We also investigated the pharmacophore of these GGPPSase inhibitors using the Catalyst method. The results demonstrated that Catalyst predicted the pIC50 values for the nine test set compounds with an rms error of 0.28 (R2 between experimental and predicted activity of 0.948).