Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we ...report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.
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Staphylococcus aureus produces a golden carotenoid virulence factor called staphyloxanthin (STX), and we report here the inhibition of the enzyme, dehydrosqualene synthase (CrtM), responsible for the ...first committed step in STX biosynthesis. The most active compounds are halogen-substituted phosphonosulfonates, with K i values as low as 5 nM against the enzyme and IC50 values for STX inhibition in S. aureus as low as 11 nM. There is, however, only a poor correlation (R 2 = 0.27) between enzyme and cell pIC50 (= −log10 IC50) values. The ability to predict cell from enzyme data improves considerably (to R 2 = 0.72) with addition of two more descriptors. We also investigated the activity of these compounds against human squalene synthase (SQS), as a counterscreen, finding several potent STX biosynthesis inhibitors with essentially no squalene synthase activity. These results open up the way to developing potent and selective inhibitors of an important virulence factor in S. aureus, a major human pathogen.
Chain gang: Lipophilic pyridinium bisphosphonates containing long alkyl chains (see picture, P red, N blue, C orange, H gray) stimulate human γδ T cells expressing the Vγ2Vδ2 T cell receptor. ...Stimulation with such compounds is more potent than with zoledronate, which is active against breast and prostate cancer. The lipophilic bisphosphonates bind poorly to bone and are thus less likely to cause side effects associated with bisphosphonates in clinical use.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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 13C, 15N, and 31P 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.
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We screened 26 bisphosphonates against a farnesyl diphosphate synthase from Plasmodium vivax, finding a poor correlation between enzyme and cell growth inhibition (R 2 = 0.06). To better predict cell ...activity data, we then used a combinatorial descriptor search in which pIC50(cell) = a pIC50(enzyme) + bB + cC + d, where B and C are descriptors (such as SlogP), and a−d are coefficients. R 2 increased from 0.01 to 0.74 (for a leave-two-out test set of 26 predictions). The method was then further validated using data for nine other systems, including bacterial, viral, and mammalian cell systems. On average, experimental/predicted cell pIC50 correlations increased from R 2 = 0.28 (for an enzyme-only test set) to 0.70 (for enzyme plus two descriptor test set predictions), while predictions based on scrambled cell activity had no predictive value (R 2 = 0.13). These results are of interest since they represent a general way to predict cell from enzyme inhibition data, with in three cases, R 2 values increasing from ∼0.02 to 0.72.
We report the results of a solid-state NMR and quantum chemical investigation of the 13Cγ 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 (R 2 = 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 φ, ψ, χ1, or χ2 torsion angles, unlike the results seen with other amino acids. There is, however, a linear relation between computed Cγ 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 π-electron density and 13C shifts for classical 4n + 2 (n = 0, 1, 2) π-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 π electron density results in an increase in Cγ atomic charge and increased shielding (of σ11 and σ22, perpendicular to the π 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 Cγ 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.
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