Bisphosphonate-Generated ATP-Analogs Inhibit Cell Signaling Pathways

Bisphosphonates are a major class of drugs used to treat osteoporosis, Paget’s disease, and cancer. They have been proposed to act by inhibiting one or more targets including protein prenylation, the epidermal growth factor receptor, or the adenine nucleotide translocase. Inhibition of the latter is due to formation in cells of analogs of ATP: the isopentenyl ester of ATP (ApppI) or an AppXp-type analog of ATP, such as AMP-clodronate (AppCCl₂p). We screened both ApppI as well as AppCCl₂p against a panel of 369 kinases finding potent inhibition of some tyrosine kinases by AppCCl₂p, attributable to formation of a strong hydrogen bond between tyrosine and the terminal phosphonate. We then synthesized bisphosphonate preprodrugs that are converted in cells to other ATP-analogs, finding low nM kinase inhibitors that inhibited cell signaling pathways. These results help clarify our understanding of the mechanisms of action of bisphosphonates, potentially opening up new routes to the development of bone resorption, anticancer, and anti-inflammatory drug leads

Catalytic Role of Conserved Asparagine, Glutamine, Serine, and Tyrosine Residues in Isoprenoid Biosynthesis Enzymes

We report the results of an investigation into the catalytic role of highly conserved amide-containing (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 analogue dimethylallyl (<i>S</i>)-thiolodiphosphate (DMASPP). The two molecules have diphosphate group orientations similar to those seen in other ζ-fold (<i>cis</i> head-to-tail and head-to-middle) prenyltransferases, with one diphosphate moiety forming a bidentate chelate with Mg²⁺ in the so-called S1 site (which is typically the allylic binding site in ζ-fold proteins) and the second diphosphate binding to Mg²⁺ 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-containing (Asn, Gln) and OH-containing (Tyr) residues seen in many “head-to-head” prenyltransferases such as squalene synthase 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 Mg²⁺ can act as a general (imine-like) base while Gln, Tyr, and H₂O 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, and H₂O) act as proton shuttles, complementing the more familiar roles of acidic groups (in Asp and Glu), which bind to Mg²⁺, and basic groups (primarily Arg), which bind to diphosphates, in isoprenoid biosynthesis

Structures of Trypanosome Vacuolar Soluble Pyrophosphatases: Antiparasitic Drug Targets

Trypanosomatid parasites are the causative agents of many neglected tropical diseases, including the leishmaniases, Chagas disease, and human African trypanosomiasis. They exploit unusual vacuolar soluble pyrophosphatases (VSPs), absent in humans, for cell growth and virulence and, as such, are drug targets. Here, we report the crystal structures of VSP1s from Trypanosoma cruzi and T. brucei, together with that of the T. cruzi protein bound to a bisphosphonate inhibitor. Both VSP1s form a hybrid structure containing an (N-terminal) EF-hand domain fused to a (C-terminal) pyrophosphatase domain. The two domains are connected via an extended loop of about 17 residues. Crystallographic analysis and size exclusion chromatography indicate that the VSP1s form tetramers containing head-to-tail dimers. Phosphate and diphosphate ligands bind in the PPase substrate-binding pocket and interact with several conserved residues, and a bisphosphonate inhibitor (BPH-1260) binds to the same site. On the basis of Cytoscape and other bioinformatics analyses, it is apparent that similar folds will be found in most if not all trypanosomatid VSP1s, including those found in insects (Angomonas deanei, Strigomonas culicis), plant pathogens (<i>Phytomonas</i> spp.), and <i>Leishmania</i> spp. Overall, the results are of general interest since they open the way to structure-based drug design for many of the neglected tropical diseases

Trapping the dynamic acyl carrier protein in fatty acid biosynthesis.

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease