Cheryl Allen

Potent Triazole Bisphosphonate Inhibitor of Geranylgeranyl Diphosphate Synthase

Studies of triazole bisphosphonates have resulted in identification of a potent inhibitor of geranylgeranyl diphosphate synthase (IC50 = 45 nM) with very good selectivity for this enzyme over farnesyl diphosphate synthase (IC50 = 28 μM). This compound also potently disrupts geranylgeranylation and induces cytotoxicity in human myeloma cells at submicromolar levels, suggesting that it may serve as a lead compound for treatment of malignancies characterized by excessive protein secretion.

Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

Multiple myeloma (MM) is characterized by the production of monoclonal protein (MP). We have shown previously that disruption of the isoprenoid biosynthetic pathway (IBP) causes a block in MP secretion through a disruption of Rab GTPase activity, leading to an enhanced unfolded protein response and subsequent apoptosis in MM cells. Autophagy is induced by cellular stressors including nutrient deprivation and ER stress. IBP inhibitors have been shown to have disparate effects on autophagy. Here we define the mechanisms underlying the differential effects of IBP inhibitors on autophagic flux in MM cells utilizing specific pharmacological inhibitors. We demonstrate that IBP inhibition induces a net increase in autophagy as a consequence of disruption of isoprenoid biosynthesis which is not recapitulated by direct geranylgeranyl transferase inhibition. IBP inhibitor-induced autophagy is a cellular defense mechanism as treatment with the autophagy inhibitor bafilomycin A1 enhances the cytotoxic effects of GGPP depletion, but not geranylgeranyl transferase inhibition. Immunofluorescence microscopy studies revealed that IBP inhibitors disrupt ER to Golgi trafficking of monoclonal light chain protein and that this protein is not a substrate for alternative degradative pathways such as aggresomes and autophagosomes. These studies support further development of specific GGTase II inhibitors as anti-myeloma agents.

Olefin Isomers of a Triazole Bisphosphonate Synergistically Inhibit Geranylgeranyl Diphosphate Synthase

The isoprenoid donor for protein geranylgeranylation reactions, geranylgeranyl diphosphate (GGDP), is the product of the enzyme GGDP synthase (GGDPS) that condenses farnesyl diphosphate (FDP) and isopentenyl pyrophosphate. GGDPS inhibition is of interest from a therapeutic perspective for multiple myeloma because we have shown that targeting Rab GTPase geranylgeranylation impairs monoclonal protein trafficking, leading to endoplasmic reticulum stress and apoptosis. We reported a series of triazole bisphosphonate GGDPS inhibitors, of which the most potent was a 3:1 mixture of homogeranyl (HG) and homoneryl (HN) isomers. Here we determined the activity of the individual olefin isomers. Enzymatic and cellular assays revealed that although HN is approximately threefold more potent than HG, HN is not more potent than the original mixture. Studies in which cells were treated with varying concentrations of each isomer alone and in different combinations revealed that the two isomers potentiate the induced-inhibition of protein geranylgeranylation when used in a 3:1 HG:HN combination. A synergistic interaction was observed between the two isomers in the GGDPS enzyme assay. These results suggested that the two isomers bind simultaneously to the enzyme but within different domains. Computational modeling studies revealed that HN is preferred at the FDP site, that HG is preferred at the GGDP site, and that both isomers may bind to the enzyme simultaneously. These studies are the first to report a set of olefin isomers that synergistically inhibit GGDPS, thus establishing a new paradigm for the future development of GGDPS inhibitors.

A new motif for inhibitors of geranylgeranyl diphosphate synthase

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon.

N-Oxide derivatives of 3-(3-pyridyl)-2-phosphonopropanoic acids as potential inhibitors of Rab geranylgeranylation

The N-oxide derivatives of [2-(3-pyridinyl)-1-hydroxyethylidene-1,1-phosphonocarboxylic acid (or PEHPC) and [2-(3-pyridinyl)-1-ethylidene-1,1-phosphonocarboxylic acid (or PEPC) have been prepared and evaluated for their activity against several enzymes which utilize isoprenoids. The parent pyridines are known inhibitors of GGTase II, but the N-oxide derivatives show no improvement in biological activity in assays with the isolated enzyme. However, the PEHPC N-oxide did induce significant accumulation of intracellular light chain in myeloma cells, consistent with inhibition of Rab geranylgeranylation.

Bishomoisoprenoid triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase

Protein geranylgeranylation reactions are dependent on the availability of geranylgeranyl diphosphate (GGDP), which serves as the isoprenoid donor. Inhibition of GGDP synthase (GGDPS) is of interest from a drug development perspective as GGDPS inhibition results in impaired protein geranylgeranylation, which in multiple myeloma, disrupts monoclonal protein trafficking and induces apoptosis. We have recently reported a series of isoprenoid triazole bisphosphonates and have demonstrated that a 3:1 mixture of homogeranyl and homoneryl isomers potently, and in a synergistic manner, inhibits GGDPS. We now present the synthesis and biological evaluation of a novel series of bishomoisoprenoid triazoles which furthers our understanding of the structure-function relationship of this class. These studies demonstrate the importance of chain length and olefin stereochemistry on inhibitory activity.

Novel tropolones induce the unfolded protein response pathway and apoptosis in multiple myeloma cells

Tropolones are small organic compounds with metal-directing moieties. Tropolones inhibit the proliferation of cancer cell lines, possibly through their effects on metalloenzymes such as select histone deacetylases (HDACs). Pan-HDAC inhibitors are therapeutically beneficial in the treatment of multiple myeloma, however there is interest in the use of more selective HDAC inhibitor therapy to minimize adverse side effects. We hypothesized that tropolones might have anti-myeloma activities. To this end, a series of novel α-substituted tropolones were evaluated for effects on multiple myeloma cells. While all tested tropolones showed some level of cytotoxicity, MO-OH-Nap had consistently low IC50 values between 1-11 μM in all three cell lines tested and was used for subsequent experiments. MO-OH-Nap was found to induce apoptosis in a concentration-dependent manner. Time course experiments demonstrated that MO-OH-Nap promotes caspase cleavage in a time frame that was distinct from the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Furthermore, MO-OH-Nap- and SAHA-treated cells possess unique gene expression patterns, suggesting they promote apoptosis via different mechanisms. In particular, MO-OH-Nap increases the expression of markers associated with endoplasmic reticulum stress and the unfolded protein response. Synergistic cytotoxic effects were observed when cells were treated with the combination of MO-OH-Nap and the proteasome inhibitor bortezomib. However, treatment with MO-OH-Nap did not abrogate the bortezomib-induced increase in aggresomes, consistent with an HDAC6-independent mechanism for the observed synergy. Collectively, these finding support further investigation into the usefulness of α-substituted tropolones as anti-myeloma agents.

Abstract 4768: Determination of Rab GTPase-mediated pathways critical for the antimyeloma activity of Rab GGTase inhibitors

Multiple myeloma (MM) is a plasma cell malignancy characterized by the production of high levels of monoclonal protein (MP). This leads to an increased protein folding burden in the endoplasmic reticulum (ER) in MM cells, resulting in a constitutively upregulated unfolded protein response (UPR) and a lower threshold for ER stress-mediated apoptosis. We have previously shown that inhibitors of Rab GGTase, the enzyme responsible for Rab GTPase geranylgeranylation, disrupt MP trafficking in MM cells, leading to an accumulation of MP in the ER and induction of the UPR and apoptosis. There are over 60 different Rab GTPases in mammalian cells involved in regulating a wide range of membrane trafficking events including exocytic, endocytic, and lysosomal pathways. In this study, we aimed to further define how disruption of RabGTPase function in MM cells leads to apoptosis by knocking down individual Rab proteins. Given the accumulation of MP and induction of UPR, our initial focus was on secretory Rabs. Knockdown of Rab1A/B (>50%) using siRNA in RPMI 8226 cells lead to moderate increases in intracellular lambda light chain in Rab1A/B knockdowns after 72 hours (∼120-140% of control levels by ELISA). However, despite the increase in intracellular MP, no increases in UPR or apoptosis markers were seen by western blot analysis or annexin-V/propidium iodide flow cytometry. Extended time course experiments (up to 120 hours) also did not exhibit any increase in UPR or apoptosis. Knockdown of Rab6A (>60%), another RabGTPase associated with the secretory pathway, did not lead to increases in intracellular lambda light chain or induction of apoptosis. These results suggest that although individual knockdown of Rab1 is sufficient to partially disrupt MP trafficking, either more complete knockdown of the individual Rabs or knockdown of multiple Rabs is necessary for induction of the UPR and apoptosis. Additional optimization of the knockdowns will be performed in order to verify these results. These studies support the further development of Rab GGTase inhibitors that globally effect Rab GTPase geranylgeranylation as a novel anti-myeloma strategy. Citation Format: Kaitlyn M. Dykstra, Cheryl L. Allen, Sarah A. Holstein. Determination of Rab GTPase-mediated pathways critical for the antimyeloma activity of Rab GGTase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4768.