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Rodney Croteau - One of the best experts on this subject based on the ideXlab platform.

  • Abietadiene synthase catalysis: conserved residues involved in protonation-initiated cyclization of Geranylgeranyl Diphosphate to (+)-copalyl Diphosphate.
    Biochemistry, 2002
    Co-Authors: Reuben J. Peters, Rodney Croteau
    Abstract:

    Abietadiene synthase catalyzes two sequential, mechanistically distinct cyclization reactions in the formation of a mixture of abietadiene double bond isomers as the committed step in resin acid biosynthesis. Each reaction is carried out at a separate active site residing in a structurally distinct domain, and the reactions are kinetically separable. The first cyclization reaction is initiated by protonation of the terminal double bond of the universal diterpene precursor, Geranylgeranyl Diphosphate. The pH dependence of the overall reaction is consistent with an acid-base catalytic mechanism, and a divalent metal ion plays a role in this reaction probably by binding the Diphosphate moiety to assist in positioning the substrate for catalysis. A putative active site for the protonation-initiated cyclization was defined by modeling abietadiene synthase and locating the DXDD motif previously shown to be involved in this reaction. A number of charged and aromatic residues, which are highly conserved in mechanistically related diterpene cyclases, line the putative active site. Alanine substitutions were made for each of these residues, as were asparagine and glutamate substitutions for the aspartates of the DXDD motif. Kinetic evaluation confirmed the involvement of most of the targeted residues in the reaction, and analysis of mutational effects on the pH-activity profile and affinity for a transition state analogue suggested specific roles for several of these residues in catalyzing the cyclization of Geranylgeranyl Diphosphate to (+)-copalyl Diphosphate. A functional role was also suggested for the cryptic insertional element found in abietadiene synthase and other diterpene synthases that carry out similar protonation-initiated cyclizations.

  • interaction with the small subunit of geranyl Diphosphate synthase modifies the chain length specificity of Geranylgeranyl Diphosphate synthase to produce geranyl Diphosphate
    Journal of Biological Chemistry, 2002
    Co-Authors: Charles Burke, Rodney Croteau
    Abstract:

    Abstract Geranyl Diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl Diphosphate synthase and Geranylgeranyl Diphosphate synthase, that catalyzes the specific formation, from C5 units, of the respective C10, C15, and C20 precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl Diphosphate synthase and Geranylgeranyl Diphosphate synthase, which are homodimers, geranyl Diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56–75%) with Geranylgeranyl Diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl Diphosphate synthases; yet it is absolutely required for geranyl Diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl Diphosphate synthase small subunit with the phylogenetically distant Geranylgeranyl Diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl Diphosphate as product in each case. These results indicate that the geranyl Diphosphate synthase small subunit is capable of modifying the chain length specificity of Geranylgeranyl Diphosphate synthase (but not, apparently, farnesyl Diphosphate synthase) to favor the production of C10 chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl Diphosphate synthase and Geranylgeranyl Diphosphate synthase.

  • abietadiene synthase catalysis conserved residues involved in protonation initiated cyclization of Geranylgeranyl Diphosphate to copalyl Diphosphate
    Biochemistry, 2002
    Co-Authors: Reuben J. Peters, Rodney Croteau
    Abstract:

    Abietadiene synthase catalyzes two sequential, mechanistically distinct cyclization reactions in the formation of a mixture of abietadiene double bond isomers as the committed step in resin acid biosynthesis. Each reaction is carried out at a separate active site residing in a structurally distinct domain, and the reactions are kinetically separable. The first cyclization reaction is initiated by protonation of the terminal double bond of the universal diterpene precursor, Geranylgeranyl Diphosphate. The pH dependence of the overall reaction is consistent with an acid-base catalytic mechanism, and a divalent metal ion plays a role in this reaction probably by binding the Diphosphate moiety to assist in positioning the substrate for catalysis. A putative active site for the protonation-initiated cyclization was defined by modeling abietadiene synthase and locating the DXDD motif previously shown to be involved in this reaction. A number of charged and aromatic residues, which are highly conserved in mechanistically related diterpene cyclases, line the putative active site. Alanine substitutions were made for each of these residues, as were asparagine and glutamate substitutions for the aspartates of the DXDD motif. Kinetic evaluation confirmed the involvement of most of the targeted residues in the reaction, and analysis of mutational effects on the pH-activity profile and affinity for a transition state analogue suggested specific roles for several of these residues in catalyzing the cyclization of Geranylgeranyl Diphosphate to (+)-copalyl Diphosphate. A functional role was also suggested for the cryptic insertional element found in abietadiene synthase and other diterpene synthases that carry out similar protonation-initiated cyclizations.

  • Cloning and Functional Expression of a cDNA Encoding Geranylgeranyl Diphosphate Synthase fromTaxus canadensisand Assessment of the Role of this Prenyltransferase in Cells Induced for Taxol Production
    Archives of biochemistry and biophysics, 1998
    Co-Authors: Jerry Hefner, Raymond E.b. Ketchum, Rodney Croteau
    Abstract:

    Geranylgeranyl Diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-induced Taxus canadensis suspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an induced T. canadensis cell library. The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce Geranylgeranyl Diphosphate from farnesyl Diphosphate and [4-14C]isopentenyl Diphosphate and was subjected to preliminary kinetic characterization. The deduced amino acid sequence of this gymnosperm Geranylgeranyl Diphosphate synthase (393 residues) resembles those of Geranylgeranyl Diphosphate synthases of angiosperm origin, except for the 90-100 N-terminal residues that correspond to the plastidial transit peptide. The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative "mature proteins" from which the transit peptide was deleted, were transformed into a yeast mutant defective for the beta-subunit of type II Geranylgeranyl transferase. Under conditions of regulated expression, both the full-length construct and the longest of the truncations (at Phe 99) were able to complement the mutant. However, when these two constructs were overexpressed in a wild-type yeast strain, they were apparently toxic, most probably due to depletion of endogenous farnesyl Diphosphate as the cosubstrate for the Geranylgeranyl Diphosphate synthase reaction. In vitro activity of the corresponding recombinant enzymes paralleled the expression level of the constructs as determined by SDS-PAGE analysis of the appropriate proteins of predicted size, and was correlated with toxicity in the wild-type yeast strain and with ability to complement the mutant strain. Results from the analysis of Geranylgeranyl Diphosphate synthase activity levels and measurement of the corresponding steady-state mRNA levels during the time course of Taxol production in induced T. canadensis suspension cell cultures, and comparison to similar data for activity and message levels for taxadiene synthase, the committed step of the pathway, indicated that for each enzyme both the level of corresponding message and catalytic activity rapidly increased after methyl jasmonate induction.

  • Stereospecific intramolecular proton transfer in the cyclization of Geranylgeranyl Diphosphate to (–)-abietadiene catalyzed by recombinant cyclase from grand fir (Abies grandis)
    Chemical Communications, 1998
    Co-Authors: Matthew M. Ravn, Robert M Coates, Reinhard Jetter, Rodney Croteau
    Abstract:

    The cyclization–rearrangement of deuterated Geranylgeranyl Diphosphate (GGPP) and (+)-copalyl Diphosphate (CPP) catalyzed by recombinant (–)-abietadiene synthase from grand fir proceeds with intramolecular proton transfer from C-19 of GGPP, and from the C-17 pro-E position of CPP, to form the C-16 pro-S methyl group of (–)-abietadiene.

Tokuzo Nishino - One of the best experts on this subject based on the ideXlab platform.

  • Effect of mutagenesis at the region upstream from the G(Q/E) motif of three types of Geranylgeranyl Diphosphate synthase on product chain-length.
    Journal of bioscience and bioengineering, 2009
    Co-Authors: Motoyoshi Noike, Tokuzo Nishino, Takashi Katagiri, Toru Nakayama, Hisashi Hemmi
    Abstract:

    (All-E) Geranylgeranyl Diphosphate synthases have been classified into three types based on the characteristic sequences around the first aspartate rich motif, which is highly conserved among the enzymes. In type I Geranylgeranyl Diphosphate synthases, which consist of archaeal enzymes, a bulky amino acid residue at the 5th position upstream from the motif plays a main role in the product determination, by blocking further elongation of prenyl chain as the bottom of the reaction cavity. On the other hand, type III Geranylgeranyl Diphosphate synthases, which consist of the enzymes from eukaryotes except for plants, use a bulky amino acid residue at the 2nd position upstream from the conserved G(Q/E) motif for product chain-length determination. Thus we introduced mutations into the region upstream from the G(Q/E) motif of Geranylgeranyl Diphosphate synthases of the three different types to confirm the importance of the region for the product chain-length determination. The results of the mutational analyses indicated that not only the 2nd but also the 3rd position upstream from the G(Q/E) motif is involved in the product chain-length determination mechanism in types I and III Geranylgeranyl Diphosphate synthases, while the amino acid substitution in this region did not affect the chain-length of the products of type II Geranylgeranyl Diphosphate synthase, which consist of the enzymes from bacteria and plants. The region upstream from the G(Q/E) motif possibly contributes to the product determination in the wide range of Geranylgeranyl Diphosphate synthases, as well as that around the first aspartate rich motif.

  • The product chain length determination mechanism of type II Geranylgeranyl Diphosphate synthase requires subunit interaction.
    The FEBS journal, 2008
    Co-Authors: Motoyoshi Noike, Tokuzo Nishino, Takashi Katagiri, Toru Nakayama, Tanetoshi Koyama, Hisashi Hemmi
    Abstract:

    The product chain length determination mechanism of type II Geranylgeranyl Diphosphate synthase from the bacterium, Pantoea ananatis, was studied. In most types of short-chain (all-E) prenyl Diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate-rich motif play a primary role in the product determination mechanism. However, type II Geranylgeranyl Diphosphate synthase lacks such bulky amino acids at these positions. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III Geranylgeranyl Diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the α-helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II Geranylgeranyl Diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II Geranylgeranyl Diphosphate synthase.

  • an alternative mechanism of product chain length determination in type iii Geranylgeranyl Diphosphate synthase
    FEBS Journal, 2003
    Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, Tokuzo Nishino
    Abstract:

    (All-E) prenyl Diphosphate synthases catalyze the consecutive condensation of isopentenyl Diphosphates with allylic prenyl Diphosphates, producing products with various chain-lengths that are unique for each enzyme. Some short-chain (all-E) prenyl Diphosphate synthases, i.e. farnesyl Diphosphate synthases and Geranylgeranyl Diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain-length of the product. However, among these enzymes, which are classified into several types based on the possessive patterns of such characteristics, type III Geranylgeranyl Diphosphate synthases, which consist of enzymes from eukaryotes (excepting plants), lack these features. In this study, we report that mutagenesis at the second position before the conserved G(Q/E) motif, which is distant from the well-studied region, affects the chain-length of the product for a type III Geranylgeranyl Diphosphate synthase from Saccharomyces cerevisiae. This clearly suggests that a novel mechanism is operative in the product determination for this type of enzyme. We also show herein that mutagenesis at the corresponding position of an archaeal medium-chain enzyme also alters its product specificity. These results provide valuable information on the molecular evolution of (all-E) prenyl Diphosphate synthases.

  • An alternative mechanism of product chain‐length determination in type III Geranylgeranyl Diphosphate synthase
    European journal of biochemistry, 2003
    Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, Tokuzo Nishino
    Abstract:

    (All-E) prenyl Diphosphate synthases catalyze the consecutive condensation of isopentenyl Diphosphates with allylic prenyl Diphosphates, producing products with various chain-lengths that are unique for each enzyme. Some short-chain (all-E) prenyl Diphosphate synthases, i.e. farnesyl Diphosphate synthases and Geranylgeranyl Diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain-length of the product. However, among these enzymes, which are classified into several types based on the possessive patterns of such characteristics, type III Geranylgeranyl Diphosphate synthases, which consist of enzymes from eukaryotes (excepting plants), lack these features. In this study, we report that mutagenesis at the second position before the conserved G(Q/E) motif, which is distant from the well-studied region, affects the chain-length of the product for a type III Geranylgeranyl Diphosphate synthase from Saccharomyces cerevisiae. This clearly suggests that a novel mechanism is operative in the product determination for this type of enzyme. We also show herein that mutagenesis at the corresponding position of an archaeal medium-chain enzyme also alters its product specificity. These results provide valuable information on the molecular evolution of (all-E) prenyl Diphosphate synthases.

  • The role of histidine‐114 of Sulfolobus acidocaldarius Geranylgeranyl Diphosphate synthase in chain‐length determination
    FEBS letters, 2000
    Co-Authors: Kazutake Hirooka, Hisashi Hemmi, Tatsuya Kato, Jun-ichiro Matsu-ura, Tokuzo Nishino
    Abstract:

    Abstract Sulfolobus acidocaldarius Geranylgeranyl Diphosphate synthase yields (all-E)-C20 prenyl Diphosphate as a final product. The three-dimensional model of the enzyme suggested that removing two bulky residues at 77 and 114 would allow additional prenyl-chain elongation. To test this, we examined several mutants with substitutions at 77 and/or 114. As a result, the mutants, F77G, F77G and H114A, F77G and H114G, H114A, and H114G gave C30, C45, C50, C30 and C40 as the main long product, respectively. These observations indicate that histidine-114 plays a crucial role in chain-length determination along with phenylalanine-77.

Hisashi Hemmi - One of the best experts on this subject based on the ideXlab platform.

  • Effect of mutagenesis at the region upstream from the G(Q/E) motif of three types of Geranylgeranyl Diphosphate synthase on product chain-length.
    Journal of bioscience and bioengineering, 2009
    Co-Authors: Motoyoshi Noike, Tokuzo Nishino, Takashi Katagiri, Toru Nakayama, Hisashi Hemmi
    Abstract:

    (All-E) Geranylgeranyl Diphosphate synthases have been classified into three types based on the characteristic sequences around the first aspartate rich motif, which is highly conserved among the enzymes. In type I Geranylgeranyl Diphosphate synthases, which consist of archaeal enzymes, a bulky amino acid residue at the 5th position upstream from the motif plays a main role in the product determination, by blocking further elongation of prenyl chain as the bottom of the reaction cavity. On the other hand, type III Geranylgeranyl Diphosphate synthases, which consist of the enzymes from eukaryotes except for plants, use a bulky amino acid residue at the 2nd position upstream from the conserved G(Q/E) motif for product chain-length determination. Thus we introduced mutations into the region upstream from the G(Q/E) motif of Geranylgeranyl Diphosphate synthases of the three different types to confirm the importance of the region for the product chain-length determination. The results of the mutational analyses indicated that not only the 2nd but also the 3rd position upstream from the G(Q/E) motif is involved in the product chain-length determination mechanism in types I and III Geranylgeranyl Diphosphate synthases, while the amino acid substitution in this region did not affect the chain-length of the products of type II Geranylgeranyl Diphosphate synthase, which consist of the enzymes from bacteria and plants. The region upstream from the G(Q/E) motif possibly contributes to the product determination in the wide range of Geranylgeranyl Diphosphate synthases, as well as that around the first aspartate rich motif.

  • The product chain length determination mechanism of type II Geranylgeranyl Diphosphate synthase requires subunit interaction.
    The FEBS journal, 2008
    Co-Authors: Motoyoshi Noike, Tokuzo Nishino, Takashi Katagiri, Toru Nakayama, Tanetoshi Koyama, Hisashi Hemmi
    Abstract:

    The product chain length determination mechanism of type II Geranylgeranyl Diphosphate synthase from the bacterium, Pantoea ananatis, was studied. In most types of short-chain (all-E) prenyl Diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate-rich motif play a primary role in the product determination mechanism. However, type II Geranylgeranyl Diphosphate synthase lacks such bulky amino acids at these positions. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III Geranylgeranyl Diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the α-helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II Geranylgeranyl Diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II Geranylgeranyl Diphosphate synthase.

  • Specific Partial Reduction of Geranylgeranyl Diphosphate by an Enzyme from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius Yields a Reactive Prenyl Donor, Not a Dead-End Product
    Journal of bacteriology, 2008
    Co-Authors: Sho Sato, Motomichi Murakami, Tohru Yoshimura, Hisashi Hemmi
    Abstract:

    Geranylgeranyl reductase from Sulfolobus acidocaldarius was shown to catalyze the reduction of Geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when Geranylgeranyl Diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from Geranylgeranyl Diphosphate. Thus, the reaction product was shown to be phytyl Diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl Diphosphate.

  • an alternative mechanism of product chain length determination in type iii Geranylgeranyl Diphosphate synthase
    FEBS Journal, 2003
    Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, Tokuzo Nishino
    Abstract:

    (All-E) prenyl Diphosphate synthases catalyze the consecutive condensation of isopentenyl Diphosphates with allylic prenyl Diphosphates, producing products with various chain-lengths that are unique for each enzyme. Some short-chain (all-E) prenyl Diphosphate synthases, i.e. farnesyl Diphosphate synthases and Geranylgeranyl Diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain-length of the product. However, among these enzymes, which are classified into several types based on the possessive patterns of such characteristics, type III Geranylgeranyl Diphosphate synthases, which consist of enzymes from eukaryotes (excepting plants), lack these features. In this study, we report that mutagenesis at the second position before the conserved G(Q/E) motif, which is distant from the well-studied region, affects the chain-length of the product for a type III Geranylgeranyl Diphosphate synthase from Saccharomyces cerevisiae. This clearly suggests that a novel mechanism is operative in the product determination for this type of enzyme. We also show herein that mutagenesis at the corresponding position of an archaeal medium-chain enzyme also alters its product specificity. These results provide valuable information on the molecular evolution of (all-E) prenyl Diphosphate synthases.

  • An alternative mechanism of product chain‐length determination in type III Geranylgeranyl Diphosphate synthase
    European journal of biochemistry, 2003
    Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, Tokuzo Nishino
    Abstract:

    (All-E) prenyl Diphosphate synthases catalyze the consecutive condensation of isopentenyl Diphosphates with allylic prenyl Diphosphates, producing products with various chain-lengths that are unique for each enzyme. Some short-chain (all-E) prenyl Diphosphate synthases, i.e. farnesyl Diphosphate synthases and Geranylgeranyl Diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain-length of the product. However, among these enzymes, which are classified into several types based on the possessive patterns of such characteristics, type III Geranylgeranyl Diphosphate synthases, which consist of enzymes from eukaryotes (excepting plants), lack these features. In this study, we report that mutagenesis at the second position before the conserved G(Q/E) motif, which is distant from the well-studied region, affects the chain-length of the product for a type III Geranylgeranyl Diphosphate synthase from Saccharomyces cerevisiae. This clearly suggests that a novel mechanism is operative in the product determination for this type of enzyme. We also show herein that mutagenesis at the corresponding position of an archaeal medium-chain enzyme also alters its product specificity. These results provide valuable information on the molecular evolution of (all-E) prenyl Diphosphate synthases.

Sarah A. Holstein - One of the best experts on this subject based on the ideXlab platform.

  • Amides as bioisosteres of triazole-based Geranylgeranyl Diphosphate synthase inhibitors
    Bioorganic & medicinal chemistry, 2020
    Co-Authors: Daniel B. Goetz, David F. Wiemer, Michelle L. Varney, Sarah A. Holstein
    Abstract:

    Geranylgeranyl Diphosphate synthase (GGDPS) inhibitors are of potential therapeutic interest as a consequence of their activity against the bone marrow cancer multiple myeloma. A series of bisphosphonates linked to an isoprenoid tail through an amide linkage has been prepared and tested for the ability to inhibit GGDPS in enzyme and cell-based assays. The amides were designed as analogues to triazole-based GGDPS inhibitors. Several of the new compounds show GGDPS inhibitory activity in both enzyme and cell assays, with potency dependent on chain length and olefin stereochemistry.

  • In Vivo Evaluation of Isoprenoid Triazole Bisphosphonate Inhibitors of Geranylgeranyl Diphosphate Synthase: Impact of Olefin Stereochemistry on Toxicity and Biodistribution
    The Journal of pharmacology and experimental therapeutics, 2019
    Co-Authors: Staci L. Haney, Michelle L. Varney, Yashpal S. Chhonker, Geoffrey A. Talmon, Lynette M. Smith, Daryl J. Murry, Sarah A. Holstein
    Abstract:

    The enzyme Geranylgeranyl Diphosphate synthase (GGDPS) synthesizes the 20-carbon isoprenoid Geranylgeranyl Diphosphate (GGPP) which is used in Geranylgeranylation reactions. In multiple myeloma (MM) cells, we have demonstrated that GGDPS inhibitors disrupt Rab Geranylgeranylation, leading to inhibition of monoclonal protein trafficking, induction of the unfolded protein response pathway (UPR) and apoptosis. We have previously reported preclinical studies with the GGDPS inhibitor VSW1198, which is a mixture of homogeranyl/homoneryl triazole bisphosphonates. Additional structure-function efforts have led to the development of the α-methylated derivatives RAM2093 (homogeranyl) and RAM2061 (homoneryl). As little is known regarding the impact of olefin stereochemistry on drug properties in vivo, we pursued additional preclinical evaluation of RAM2093 and RAM2061. In MM cell lines, both isomers induce activation of UPR/apoptotic markers in a concentration-dependent manner and with similar potency. Single dose testing in CD-1 mice identified a maximum tolerated dose of 0.5 mg/kg IV for RAM2061 and 0.3 mg/kg for RAM2093. Liver toxicity was the primary barrier to dose escalation for both compounds. Disruption of Geranylgeranylation in vivo was confirmed following multi-dose administration of either compound. Pharmacokinetic studies revealed plasma terminal half-lives of 29.2 ± 6 h (RAM2061) and 22.1 ± 4 h (RAM2093). Relative to RAM2061, RAM2093 levels were significantly higher in liver tissue but not in other tissues. Using MM.1S flank xenografts we observed a significant reduction in tumor growth in mice treated with RAM2061 relative to controls. Collectively, these studies reveal olefin stereochemistry-dependent effects on GGDPS inhibitor biodistribution and confirm the in vivo efficacy of this novel therapeutic approach. SIGNIFICANCE STATEMENT These studies reveal olefin stereochemistry-dependent effects on the in vivo properties of two novel triazole bisphosphonate inhibitors of Geranylgeranyl Diphosphate synthase and demonstrate the therapeutic potential of this class of inhibitors for the treatment of multiple myeloma.

  • Preclinical investigation of a potent Geranylgeranyl Diphosphate synthase inhibitor
    Investigational New Drugs, 2018
    Co-Authors: Staci L. Haney, Michelle L. Varney, Yashpal S. Chhonker, Geoffrey A. Talmon, Daryl J. Murry, Sarah A. Holstein
    Abstract:

    Geranylgeranyl Diphosphate synthase (GGDPS) is the enzyme in the isoprenoid biosynthesis pathway that catalyzes the synthesis of the 20-carbon isoprenoid GGPP, which serves as the isoprenoid donor for protein Geranylgeranylation reactions. Rab proteins mediate vesicle trafficking within the cell and their activity is dependent on Geranylgeranylation. Our prior work has demonstrated that agents that disrupt Rab Geranylgeranylation disrupt monoclonal protein trafficking in myeloma cells, resulting in induction of the unfolded protein response pathway and apoptosis. VSW1198 is a potent GGDPS inhibitor with measurable cellular activity at concentrations as low as 30 nM. Due to its potent activity against myeloma cells in vitro, we were interested in evaluating the toxicology profile, pharmacokinetic (PK) profile, tissue distribution pattern and metabolic stability of VSW1198 in preparation for in vivo efficacy studies. Single dose testing via IV administration in CD-1 mice revealed a maximum tolerated dose of 0.5 mg/kg. Doses ≥1 mg/kg resulted in liver toxicity that peaked around 6–7 days post-injection. Disruption of protein Geranylgeranylation following repeat dosing of VSW1198 was confirmed via immunoblot analysis of unmodified Rap1a in multiple organs. The PK studies revealed a half-life of 47.7 ± 7.4 h. VSW1198 was present in all tested tissues with the highest levels in the liver. In both human liver microsomes and mouse S9 studies VSW1198 showed complete stability, suggesting no phase I or phase II metabolism. In summary, these studies demonstrate systemic distribution, on-target disruption of protein Geranylgeranylation, and metabolic stability of a potent GGDPS inhibitor VSW1198 and form the basis for future efficacy studies in mouse models of myeloma.

  • α-Methylation enhances the potency of isoprenoid triazole bisphosphonates as Geranylgeranyl Diphosphate synthase inhibitors.
    Bioorganic & medicinal chemistry, 2017
    Co-Authors: Robert A. Matthiesen, David F. Wiemer, Michelle L. Varney, Alex S. Rier, Sarah A. Holstein
    Abstract:

    Abstract Disruption of protein Geranylgeranylation via inhibition of Geranylgeranyl Diphosphate synthase (GGDPS) represents a novel therapeutic strategy for a variety of malignancies, especially those characterized by excessive protein secretion such as multiple myeloma. Our work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. Here we present the synthesis and biological evaluation of a new series of isoprenoid triazoles modified by incorporation of a methyl group at the α-carbon. These studies reveal that incorporation of an α-methyl substituent enhances the potency of these compounds as GGDPS inhibitors, and, in the case of the homogeranyl/homoneryl series, abrogates the effects of olefin stereochemistry on inhibitory activity. The incorporation of the methyl group allowed preparation of a POM-prodrug, which displayed a 10-fold increase in cellular activity compared to the corresponding salt. These studies form the basis for future preclinical studies investigating the anti-myeloma activity of these novel α-methyl triazole bisphosphonates.

  • Recent Advances in the Development of Mammalian Geranylgeranyl Diphosphate Synthase Inhibitors.
    Molecules (Basel Switzerland), 2017
    Co-Authors: Staci L. Haney, David F. Wiemer, Veronica S. Wills, Sarah A. Holstein
    Abstract:

    The enzyme Geranylgeranyl Diphosphate synthase (GGDPS) catalyzes the synthesis of the 20-carbon isoprenoid Geranylgeranyl Diphosphate (GGPP). GGPP is the isoprenoid donor for protein Geranylgeranylation reactions catalyzed by the enzymes Geranylgeranyl transferase (GGTase) I and II. Inhibitors of GGDPS result in diminution of protein Geranylgeranylation through depletion of cellular GGPP levels, and there has been interest in GGDPS inhibitors as potential anti-cancer agents. Here we discuss recent advances in the development of GGDPS inhibitors, including insights gained by structure-function relationships, and review the preclinical data that support the continued development of this novel class of drugs.

Robert M Coates - One of the best experts on this subject based on the ideXlab platform.