Bisphosphonate Analogues Research Articles

Fluorination Influences the Bioisostery of Myo-Inositol Pyrophosphate Analogs.

Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.

Metal-polyDNA nanoparticles reconstruct osteoporotic microenvironment for enhanced osteoporosis treatment.

Current clinical approaches to osteoporosis primarily target osteoclast biology, overlooking the synergistic role of bone cells, immune cells, cytokines, and inorganic components in creating an abnormal osteoporotic microenvironment. Here, metal-polyDNA nanoparticles (Ca-polyCpG MDNs) composed of Ca2+ and ultralong single-stranded CpG sequences were developed to reconstruct the osteoporotic microenvironment and suppress osteoporosis. Ca-polyCpG MDNs can neutralize osteoclast-secreted hydrogen ions, provide calcium repletion, promote remineralization, and repair bone defects. Besides, the immune-adjuvant polyCpG in MDNs could induce the secretion of osteoclastogenesis inhibitor interleukin-12 and reduce the expression of osteoclast function effector protein to inhibit osteoclast differentiation, further reducing osteoclast-mediated bone resorption. PPi4- generated during the rolling circle amplification reaction acts as bisphosphonate analog and enhances bone targeting of Ca-polyCpG MDNs. In ovariectomized mouse and rabbit models, Ca-polyCpG MDNs prevented bone resorption and promoted bone repair by restoring the osteoporotic microenvironment, providing valuable insights into osteoporosis therapy.

Assigning the Absolute Configuration of Inositol Poly- and Pyrophosphates by NMR Using a Single Chiral Solvating Agent.

Inositol phosphates constitute a family of highly charged messenger molecules that play diverse roles in cellular processes. The various phosphorylation patterns they exhibit give rise to a vast array of different compounds. To fully comprehend the biological interconnections, the precise molecular identification of each compound is crucial. Since the myo-inositol scaffold possesses an internal mirror plane, enantiomeric pairs can be formed. Most commonly employed methods for analyzing InsPs have been geared towards resolving regioisomers, but they have not been capable of resolving enantiomers. In this study, we present a general approach for enantiomer assignment using NMR measurements. To achieve this goal, we used 31P-NMR in the presence of L-arginine amide as a chiral solvating agent, which enables the differentiation of enantiomers. Using chemically synthesized standard compounds allows for an unambiguous assignment of the enantiomers. This method was applied to highly phosphorylated inositol pyrophosphates, as well as to lowly phosphorylated inositol phosphates and bisphosphonate analogs. Our method will facilitate the assignment of biologically relevant isomers when isolating naturally occurring compounds from biological specimens.

Design of Extended Bisphosphonate-Based Coordination Polymers as Bone-Targeted Drug Delivery Systems for Breast Cancer-Induced Osteolytic Metastasis and Other Bone Therapies.

The coordination between benzene 1,4-bis(bisphosphonic acid) (BBPA), the bisphosphonate (BP) analogue of benzene 1,4-dicarboxylic acid (BDC), and bioactive metals led to the formation of extended bisphosphonate-based coordination polymers (BPCPs). Four distinct crystalline phases were obtained, namely, BBPA-Ca forms I and II, BBPA-Zn, and BBPA-Mg. Among these, BBPA-Ca forms I (7 × 9 Å2) and II (8 × 12 Å2) possess channels large enough to encapsulate 5-fluorouracil (5-FU), a drug prescribed in combination with BPs to treat breast cancer-induced osteolytic metastases (OM). Dissolution curves show a 14% release of BBPA from BBPA-Ca form II in phosphate-buffered saline, while ∼90% was released in fasted-state simulated gastric fluid. These results suggest that this material is relatively stable in neutral environments yet collapses in acidic conditions. Moreover, the phase inversion temperature method decreased the particle size of BBPA-Ca form II, resulting in nano-Ca@BBPA (∼134 d.nm). Binding assays showed a higher affinity of nano-Ca@BBPA (∼97%) to hydroxyapatite than BBPA (∼70%) and significantly higher binding than commercial BPs, zolendronic (3.0×), and risedronic (2.4×) acids after 24 h. Furthermore, both BBPA-Ca form II and nano-Ca@BBPA presented comparable drug loading and release (∼30 wt % 5-FU) relative to BDC-based CCs (UiO-66, MIL-53, and BDC-Zr) where other pharmaceutical compounds (caffeine, ibuprofen, aspirin, and α-cyano-4-hydroxycinnamic acid) have been encapsulated. Cell viability assays established that drug-loaded nano-Ca@BBPA increases the cytotoxicity of a triple-negative human breast cancer cell line (MDA-MB-231) when compared to 5-FU (%RCV = 8 ± 5 vs 75 ± 1% at a 100 μM). At the same concentration, no significant decrease in cell viability was observed for normal human osteoblast-like hFOB 1.19 cells (%RCV = 85 ± 1%). Collectively, these results demonstrate the feasibility of nano-Ca@BBPA as a potential drug delivery system (DDS), with high affinity to bone tissue, to treat bone-related diseases such as OM.

Radiolabeling, Quality Control, and Cell Binding Studies of New 99mTc-Labeled Bisphosphonates: 99mTc-Ibandronate Sodium.

Early detection of bone cancer is critical for treating symptoms, minimizing pain, and increasing overall quality of life. It is critical to develop novel radiopharmaceuticals with high labeling efficiency and stability for the diagnosis of bone cancer. This research aims to design a novel radiopharmaceutical that may be used to diagnose bone cancer. In this study, ibandronate sodium (IBD), a bisphosphonate analog, was radiolabeled with technetium-99m [99mTc] and quality control tests on the newly developed radiopharmaceutical ([99mTc]Tc-IBD) were performed using radioactive thin layer chromatography. After that, the incorporation of [99mTc]Tc-IBD into hydroxyapatite (HA) crystals and a human bone osteosarcoma cell line (U2OS) was tested. According to the results obtained, optimal radiolabeling procedure was obtained for [99mTc]Tc-IBD with 200 μg.mL-1 IBD, 20 μg stannous chloride, and 99mTc with 37 MBq radioactivity. The reaction mixture was adjusted to pH 5.5 and incubated at room temperature for 15 min. The radiochemical purity of [99mTc]Tc-IBD was found to be greater than 95% at room temperature for up to 6 h. Additionally, chromatography analysis showed >95% [99mTc]Tc-IBD complex formation with promising stability for up to 24 h in saline and up to 2 h in cell medium. The percentage binding of IBD to HA was 83.70 ± 3.67 and the logP of [99mTc]Tc-IBD was -1.1014. The radiolabeled complex exhibited a higher rate of cell incorporation to U2OS cells compared to Reduced/Hydrolyzed 99mTcO4 -. The newly produced radiopharmaceutical is very promising according to the results of in vitro cell culture, HA binding, and quality studies, and will be a step forward for further studies in nuclear medicine for bone cancer diagnostics.

Design, synthesis, molecular docking, dynamics and in vitro evaluation of novel 2- substituted-1-hydroxyethane-1, 1-bis(phosphonic acid) derivatives as human farnesyl pyrophosphate synthase inhibitors with expected anticancer activity.

Nitrogenous bisphosphonates (NBPs) are the major class of drugs that are used to treat osteoporosis. Recently, bisphosphonates (BPs) were reported to have an anticancer effect. These agents feature a high affinity that enables them to bind strongly to the human farnesyl pyrophosphate synthase enzyme. The correlation between this affinity and their anticancer effect was confirmed. To date, the use of an oxygen atom as an isosteric replacement for the electronegative nitrogen atom in NBPs has not been reported, and its ability to retain the linker length and bisphosphonate pharmacophore remains unknown. The main aim of this work was to design some isosteric bisphosphonate analogs with oxygen atoms and evaluation of their binding affinity and anticancer activity. The binding mode and stability of the designed compounds were achieved using human farnesyl pyrophosphate synthase (HFPPS) by docking and dynamic simulations. The compounds were synthesized, characterized, and screened for their anticancer activity against the breast cancer MCF-7 cell line and lung cancer A-549 cell line. The inhibitory activity of the tested compounds against HFPPS was evaluated. The compounds under investigation showed potential anticancer activity against the lung cell line with IC50 values of 41.7, 47.4, and 34.8 μg/ml in comparison to that of Risedronic acid (115 μg/ml). However, they do not exhibit potential activity against the breast cancer cell line. Compounds VII and VIII showed in vitro inhibition of human farnesyl pyrophosphate synthase with IC50 values of 82.2 and 98.8 μg/ml, respectively. Further optimization may be required in the future.

Prodrugs of pyrophosphates and bisphosphonates: disguising phosphorus oxyanions.

Pyrophosphates have important functions in living systems and thus pyrophosphate-containing molecules and their more stable bisphosphonate analogues have the potential to be used as drugs for treating many diseases including cancer and viral infections. Both pyrophosphates and bisphosphonates are polyanionic at physiological pH and, whilst this is essential for their biological activity, it also limits their use as therapeutic agents. In particular, the high negative charge density of these compounds prohibits cell entry other than by endocytosis, prevents transcellular oral absorption and causes sequestration to bone. Therefore, prodrug strategies have been developed to temporarily disguise the charges of these compounds. This review examines the various systems that have been used to mask the phosphorus-containing moieties of pyrophosphates and bisphosphonates and also illustrates the utility of such prodrugs.

The role of inhibition of osteocyte apoptosis in mediating orthodontic tooth movement and periodontal remodeling: a pilot study

BackgroundOrthodontic tooth movement (OTM) has been shown to induce osteocyte apoptosis in alveolar bone shortly after force application. However, how osteocyte apoptosis affects orthodontic tooth movement is unknown. The goal of this study was to assess the effect of inhibition of osteocyte apoptosis on osteoclastogenesis, changes in the alveolar bone density, and the magnitude of OTM using a bisphosphonate analog (IG9402), a drug that affects osteocyte and osteoblast apoptosis but does not affect osteoclasts.Material and methodsTwo sets of experiments were performed. Experiment 1 was used to specifically evaluate the effect of IG9402 on osteocyte apoptosis in the alveolar bone during 24 h of OTM. For this experiment, twelve mice were divided into two groups: group 1, saline administration + OTM24-h (n=6), and group 2, IG9402 administration + OTM24-h (n=6). The contralateral unloaded sides served as the control. The goal of experiment 2 was to evaluate the role of osteocyte apoptosis on OTM magnitude and osteoclastogenesis 10 days after OTM. Twenty mice were divided into 4 groups: group 1, saline administration without OTM (n=5); group 2, IG9402 administration without OTM (n=5); group 3, saline + OTM10-day (n=6); and group 4, IG9402 + OTM10-day (n=4). For both experiments, tooth movement was achieved using Ultra Light (25g) Sentalloy Closed Coil Springs attached between the first maxillary molar and the central incisor. Linear measurements of tooth movement and alveolar bone density (BVF) were assessed by MicroCT analysis. Cell death (or apoptosis) was assessed by terminal dUTP nick-end labeling (TUNEL) assay, while osteoclast and macrophage formation were assessed by tartrate-resistant acid phosphatase (TRAP) staining and F4/80+ immunostaining.ResultsWe found that IG9402 significantly blocked osteocyte apoptosis in alveolar bone (AB) at 24 h of OTM. At 10 days, IG9402 prevented OTM-induced loss of alveolar bone density and changed the morphology and quality of osteoclasts and macrophages, but did not significantly affect the amount of tooth movement.ConclusionOur study demonstrates that osteocyte apoptosis may play a significant role in osteoclast and macrophage formation during OTM, but does not seem to play a role in the magnitude of orthodontic tooth movement.

Structure-Function Relationships of Novel Benzimidazole-Based Inhibitors of the Isoprenoid Biosynthetic Pathway in Myeloma Cells

Malignant plasma cells are characterized by the production of monoclonal protein (MP). Our work has demonstrated that disrupting intracellular MP trafficking by targeting the geranylgeranylation of Rab GTPases results in endoplasmic reticulum stress, induction of the unfolded protein response pathway and apoptosis of myeloma cells. Rab proteins are geranylgeranylated by the enzyme geranylgeranyl transferase II (GGTase II) which uses geranylgeranyl diphosphate as the isoprenoid substrate. We have pursued the development of specific GGTase II and geranylgeranyl diphosphate synthase (GGDPS) inhibitors as potential anti-myeloma agents. A family of compounds incorporating a benzimidazole heterocycle and either a carboxyphosphonate or bisphosphonate was prepared by modification of the appropriate heterocycles. The core benzimidazole phosphonate structure was modified with substituents at either the alpha-carbon position or at the 5 and 6 positions on the benzimidazole (Figure). These compounds were subjected to in vitro enzyme assays for GGTase II and the related prenyltransferases farnesyl transferase (FTase) and GGTase I as well as the synthases farnesyl diphosphate synthase (FDPS) and GGDPS. Cellular disruption of protein prenylation was assessed via immunoblot analysis for Ras (a representative farnesylated protein) and for unmodified Rap1a (a representative geranylgeranylated protein) in human myeloma cells (RPMI-8226, MM.1S). The ability of the novel compounds to disrupt MP trafficking was determined by measuring changes in intracellular lambda light chain levels via ELISA. Confirmation of disruption of Rab geranylgeranylation was achieved by Triton X-114 lysis to obtain detergent (membrane) fractions which were subjected to Rab6 (a representative Rab protein) immunoblot analysis. These studies demonstrated that the nature of the phosphonate head group has a profound impact on enzyme specificity: the carboxyphosphonates have specific activity as GGTase II inhibitors while the bisphosphonate analogues have specific activity as FDPS inhibitors with no cross-reactivity against any of the other tested enzymes. The most potent GGTase II and FDPS inhibitors were the unsubstituted analogues. The carboxyphosphonate 1 was found to have an IC50 of 230 μM against GGTase II and cellular activity starting at 2.5 mM. This represents a two-fold improvement in potency compared with the known GGTase II inhibitor 3-PEHPC. The bisphosphonate 5 has an IC50 of 24 nM against FDPS and cellular activity at ≥ 50 μM. The addition of a methyl group at the alpha-carbon position abrogated the activity of the carboxyphosphonate (2) as a GGTase II inhibitor and the activity of the bisphosphonate (6) as a FDPS inhibitor. The addition of substituents at the 5 and 6 positions (compounds 3, 4, 7 and 8)did not improve potency compared with the parent compounds. Co-incubation with low concentrations of the HMG-CoA reductase inhibitor lovastatin (0.1-1 μM), concentrations which on their own are not sufficient to disrupt protein prenylation, potentiated the cellular activity of the lead GGTase II and FDPS inhibitors. In conclusion, these benzimidazole phosphonates represent novel classes of GGTase II and FDPS inhibitors. These studies highlight the importance of the identity of the phosphonate head-group in dictating enzyme specificity. Further studies are underway to prepare additional benzimidazole analogues in order to improve their potency, advance the understanding of the structure-function relationship, and assess the potential of these compounds as anti-myeloma agents. [Display omitted] DisclosuresWiemer:Terpenoid Therapeutics, Inc: Other: Founder. Holstein:Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy.

Supported Synthesis of Adenosine Nucleotides and Derivatives on a Benzene‐Centered Tripodal Soluble Support

AbstractThe first soluble‐phase synthesis of adenosine nucleotides including α,β and β,γ‐methylene bisphosphonate analogues on a multi‐pod support is reported. Anchoring of a 2’,3’‐protected purine nucleoside to the tripodal support via the nucleobase was successfully achieved using a microwave assisted Cu(I)‐catalyzed azide‐alkyne cycloaddition. Then, phosphorylation was performed, followed by cleavage with aqueous ammonia to provide adenine derivatives, and finally deprotection. When using benzylamine instead of ammonia, a derivative with N6‐benzylamine adenine as nucleobase was obtained. This methodology allows to access adenosine 5’‐mono, di and triphosphates, as well as various analogues of pharmacological interest in modest to good yields.

New Structural Insights Reveal an Expanded Reaction Cycle for Inositol Pyrophosphate Hydrolysis by Human DIPP1

Nudix hydrolases, an enzyme superfamily with representatives in all classes of organisms, possess a conserved catalytic sequence (Nudix motif), GX5EX7REUXEEXGU, where ‘U’ designates a hydrophobic residue. The Diphosphoinositol Polyphosphate Phosphohydrolases (DIPPs) represent one subgroup of the Nudix family. By metabolizing diphosphoinositol polyphosphates (PP-InsPs), DIPPs switch-off their signaling activities that regulate metabolic homeostasis. However, there has not been a molecular rationalization of DIPPs catalytic mechanism, substrate recognition and reaction specificity. We now provide X-ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP-InsPs, alternative PP-InsP stereoisomers, or non-hydrolysable methylene bisphosphonate analogs (“PCP-InsPs”). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V-shaped catalytic furrow with overhanging ridges constructed from flexible positively charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as “suspension cables.” We demonstrate functionality for a triglycine peptide within a β-strand which represents a non-canonical addition to the standard Nudix catalytic core structure. We describe pre-reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time-resolved analysis, we uncover a chronological sequence of DIPP1/product post-reaction states, one of which may rationalize a role for InsP6 as an inhibitor of catalytic activity. We have gained considerable structure-based insight into the reaction cycle for DIPP1-catalyzed PP-InsP hydrolysis and can use this information to design inhibitors of an enzyme with proposed roles in obesity, insulin signaling and sarcopenia.

Bisphosphonates in dentistry: Historical perspectives, adverse effects, and novel applications.

Studies of the potential role of bisphosphonates in dentistry date back to physical chemical research in the 1960s, and the genesis of the discovery of bisphosphonate pharmacology in part can be linked to some of this work. Since that time, parallel research on the effects of bisphosphonates on bone metabolism continued, while efforts in the dental field included studies of bisphosphonate effects on dental calculus, caries, and alveolar bone loss. While some utility of this drug class in the dental field was identified, leading to their experimental use in various dentrifice formulations and in some dental applications clinically, adverse effects of bisphosphonates in the jaws have also received attention. Most recently, certain bisphosphonates, particularly those with strong bone targeting properties, but limited biochemical effects (low potency bisphosphonates), are being studied as a local remedy for the concerns of adverse effects associated with other more potent members of this drug class. Additionally, low potency bisphosphonate analogs are under study as vectors to target active drugs to the mineral surfaces of the jawbones. These latter efforts have been devised for the prevention and treatment of oral problems, such as infections associated with oral surgery and implants. Advances in the utility and mechanistic understanding of the bisphosphonate class may enable additional oral therapeutic options for the management of multiple aspects of dental health.

Synthesis of 8-oxo-dGTP and its β,γ-CH2-, β,γ-CHF-, and β,γ-CF2- analogues

Three novel bisphosphonate analogues of 8-oxo-dGTP 3 in which the bridging β,γ-oxygen is replaced by a methylene, fluoromethylene or difluoromethylene group (4–6, respectively) have been synthesized from 8-oxo-dGMP 2 by reaction of its morpholine 5′-phosphoramidate 14 or preferably, its N-methylimidazole 5′-phosphoramidate 15 with tri-n-butylammonium salts of the appropriate bisphosphonic acids, 11–13. The latter method also provides a convenient new route to 3. Analogues 4–6 may be useful as mechanistic probes for the role of 3 in abnormal DNA replication and repair.

New structural insights reveal an expanded reaction cycle for inositol pyrophosphate hydrolysis by human DIPP1

Nudix hydrolases attract considerable attention for their wide range of specialized activities in all domains of life. One particular group of Nudix phosphohydrolases (DIPPs), through their metabolism of diphosphoinositol polyphosphates (PP-InsPs), regulates the actions of these polyphosphates upon bioenergetic homeostasis. In the current study, we describe, at an atomic level, hitherto unknown properties of human DIPP1.We provide X-ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP-InsPs, alternative PP-InsP stereoisomers, or non-hydrolysable methylene bisphosphonate analogs ("PCP-InsPs"). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V-shaped catalytic furrow with overhanging ridges constructed from flexible positively charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as "suspension cables." We demonstrate functionality for a triglycine peptide within a β-strand which represents a non-canonical addition to the standard Nudix catalytic core structure. We describe pre-reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time-resolved analysis, we uncover a chronological sequence of DIPP1/product post-reaction states, one of which may rationalize a role for InsP6 as an inhibitor of catalytic activity.

The efficacy and safety of bisphosphonate analogs for treatment of osteoporosis after spinal cord injury: a systematic review and meta-analysis of randomized controlled trials.

Rapid bone loss in the early stages of spinal cord injury (SCI) leads to an increased risk of osteoporotic fracture. A meta-analysis was conducted to assess the efficacy and safety of bisphosphonates for the treatment of osteoporosis after SCI. A literature search of the PubMed, EMBASE, Cochrane Library, and Web of Science databases identified nine randomized controlled trials with 206 individuals. This meta-analysis was performed using a random-effects model. The primary outcome was the percent change in bone mineral density (BMD) of the total hip, distal femur, and lumbar spine from baseline to 12 months. Bone turnover markers were secondary outcomes. The incidences of adverse events were assessed in order to evaluate safety. There were significant differences in BMD of the total hip and lumbar spine or serum C-terminal telopeptide between the bisphosphonate and control groups but no difference in adverse events. The percent change in BMD of the distal femur and serum type 1 procollagen N-terminal peptide from baseline to 12 months was not superior in the treatment groups. Osteoclast-mediated bone resorption was inhibited by bisphosphonate administration. Subgroup analyses of participants treated with zoledronate at different sites revealed a beneficial effect on BMD of the total hip and lumbar spine but not the distal femur. Early bisphosphonate administration after SCI was safe and beneficial to the BMD of the total hip and lumbar spine at 12 months.

Dissecting the activation of insulin degrading enzyme by inositol pyrophosphates and their bisphosphonate analogs.

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are densely phosphorylated eukaryotic messengers, which are involved in numerous cellular processes. To elucidate their signaling functions at the molecular level, non-hydrolyzable bisphosphonate analogs of inositol pyrophosphates, PCP-InsPs, have been instrumental. Here, an efficient synthetic strategy to obtain these analogs in unprecedented quantities is described – relying on the use of combined phosphate ester-phosphoramidite reagents. The PCP-analogs, alongside their natural counterparts, were applied to investigate their regulatory effect on insulin-degrading enzyme (IDE), using a range of biochemical, biophysical and computational methods. A unique interplay between IDE, its substrates and the PP-InsPs was uncovered, in which the PP-InsPs differentially modulated the activity of the enzyme towards short peptide substrates. Aided by molecular docking and molecular dynamics simulations, a flexible binding mode for the InsPs/PP-InsPs was identified at the anion binding site of IDE. Targeting IDE for therapeutic purposes should thus take regulation by endogenous PP-InsP metabolites into account.

Control of XPR1-dependent cellular phosphate efflux by InsP8 is an exemplar for functionally-exclusive inositol pyrophosphate signaling

Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP8 synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP8) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP8 levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP8; PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP8 to the XPR1 N-terminus (Kd = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification.

СУМІСНА ДІЯ НОВИХ ПІРАЗОЛВМІСНИХ БІСФОСФОНАТІВ І ВІТАМІНУ D3 ЗА ЕКСПЕРИМЕНТАЛЬНОГО ОСТЕОПОРОЗУ

Bisphosphonates remain one of the most effective drugs to treat diseases of the musculoskeletal system. Among a wide range of possible options, the methods of bioinformatic analysis were developed, selected and synthesized analogs of bisphosphonates, the radical of which were selected pyrazole derivatives. The development of new advanced bisphosphonate analogues has broad prospects for medical application. The study is devoted to the investigation of the combined action of synthesized nitrogen-containing bisphosphonates (pyrazole-containing analogues) and vitamin D3 in the correction of mineral metabolism disturbances in osteoporosis. The work was performed on a rat model of alimentary osteoporosis, which was characterized by the development of hypocalcemia, hypophosphatemia, decreased serum 25OHD concentration and severe bone tissue demineralization. New pyrazole-containing bisphosphonates inhibited the process of bone demineralization (resorption) and improved mineral metabolism in rats with alimentary osteoporosis with varying efficiency. The action of the investigated substances resulted in an increase in the concentration of calcium, inorganic phosphate and a decrease in the activity of alkaline phosphatase and its isoenzymes in the serum. The ash content, calcium and phosphorus level in the ash of the tibia was increased as well, along with improving osteometric characteristics of the tibia (length and thickness of the proximal epimetaphysis). The combination of pyrazole-containing bisphosphonates and vitamin D3, which normalizes the concentration of 25OHD in the serum and provides the synthesis of biologically active, hydroxylated form of cholecalciferol, has been more effective in the correction of mineral metabolism impairments in rats with alimentary osteoporosis. According to the experimental results, the combination of vitamin D3 with bisphosphonate ISP-30, as a substance with the highest biological efficiency in regulating bone remodeling, enhancing osteogenesis and inhibition of resorption, is the most promising for further research.

Novel benzimidazole phosphonates as potential inhibitors of protein prenylation

Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.

New chirally modified bisphosphonates for synthesis of individual beta,gamma-CHX-deoxynucleotide diastereomers

Individual diastereomers of CXY bisphosphonate analogues of dNTPs or NTPs are useful chemical stereoprobes to investigate interactions within the chiral active site environment of enzymes such as polymerases and kinases. We previously reported synthetic access to β,γ-CHX-dGTPs (X = F or Cl) via a bisphosphonate synthon with an (R)-methyl mandelate auxiliary and have extended this approach to dTTP and dATP analogues. As removal of the chiral auxiliary by (Pd/C) hydrogenolysis is incompatible with the cytosine heterocycle and also with X = Br, we have now designed bisphosphonate synthons using (R)-(+)-α-ethylbenzylamine or methyl (R)-(–)-phenylglycine auxiliaries and equipped with an o-nitrobenzyl ester protecting group allowing photochemical deprotection. These new synthons have made possible the first syntheses of individual dCTP and monobromo-substituted dNTP β,γ-CHX diastereomers.