Biomedical subjects

R Graham G Russell

Publications and source records attributed to R Graham G Russell.

9 recordsLinked to original sources

PubMed2007

Apomine, an inhibitor of HMG-CoA-reductase, promotes apoptosis of myeloma cells in vitro and is associated with a modulation of myeloma in vivo.

Apomine, a novel 1,1 bisphosphonate ester, increases the rate of degradation of HMG-CoA reductase, inhibiting the mevalonate pathway and thereby blocking cholesterol biosynthesis. We have investigated whether Apomine can induce myeloma cell apoptosis in vitro and modulate myeloma disease in vivo. Apomine induced a dose-dependent increase in apoptosis in NCI H929, RPMI 8226 and JJN-3 human myeloma cells. Apomine, unlike the bisphosphonate, alendronate, had no measurable effect on osteoclastic bone resorption in vitro. To investigate the effect of Apomine in vivo, 5T2MM murine myeloma cells were injected into C57BL/KaLwRij mice. After 8 weeks all animals had a serum paraprotein and were treated with Apomine (200 mg/kg), or vehicle, for 4 weeks. Animals injected with 5T2MM cells and treated with vehicle developed osteolytic bone lesions, reduced cancellous bone area, decreased bone mineral density (BMD) and increased osteoclast number. Apomine caused a decrease in serum paraprotein and a decrease in tumor burden. Apomine inhibited the development of osteolytic lesions and prevented the tumor-induced decreases in BMD. Apomine had no effect on osteoclast number in contrast to what had been seen previously with the bisphosphonate, zoledronic acid, suggesting that these are direct effects of Apomine on myeloma cells. This demonstrates that Apomine is able to promote myeloma cell apoptosis in vitro and inhibit the development of multiple myeloma and lytic bone disease in vivo. The use of bisphosphonate esters such as Apomine represents a novel therapeutic approach in the treatment of myeloma and, indirectly, the associated bone disease.

Animals↗

PubMed2006

Selective inhibition of Rab prenylation by a phosphonocarboxylate analogue of risedronate induces apoptosis, but not S-phase arrest, in human myeloma cells.

Bisphosphonates (BPs) are widely used in the treatment of osteolytic bone disease associated with multiple myeloma, and have been demonstrated to exert antitumor effects both in vitro and in vivo. However, the precise molecular mechanisms involved in the direct antitumor effects of BPs in vitro are not known. Nitrogen-containing BPs, such as risedronate (RIS), act by inhibiting protein prenylation. A phosphonocarboxylate analogue of RIS, 3-PEHPC, has previously been shown in osteoclasts and macrophages to specifically inhibit prenylation of Rab GTPases. The aim of this study was to identify the molecular targets of RIS and 3-PEHPC in human myeloma cells and to determine the cellular effects of selective inhibition of Rab prenylation by 3-PEHPC as compared to nonspecific inhibition of protein prenylation by RIS in human myeloma cells. RIS dose-dependently inhibited prenylation of both Rap1A and Rab6, whereas 3-PEHPC only inhibited Rab6 prenylation. Both RIS and 3-PEHPC dose-dependently increased apoptosis in human myeloma cells. RIS induced an accumulation of cells in the S-phase of the cell cycle, associated with inhibition of DNA replication. In contrast, 3-PEHPC did not cause cell-cycle arrest. Furthermore, geranylgeraniol could prevent inhibition of prenylation, induction of apoptosis, and cell-cycle arrest in response to RIS, but not inhibition of Rab prenylation and apoptosis induced by 3-PEHPC, consistent with specific inhibition of Rab geranylgeranyl transferase by 3-PEHPC. In conclusion, our studies demonstrate that selective inhibition of Rab prenylation induces apoptosis, but not S-phase arrest, thus identifying distinct molecular pathways that mediate the antimyeloma effect of nitrogen-containing BPs.

Alkyl and Aryl Transferases↗

PubMed2006

The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding.

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of approximately 200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis.

Crystallization↗

PubMed2006

The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs.

Osteoporosis and low bone mass are currently estimated to be a major public health risk affecting >50% of the female population over the age of 50. Because of their bone-selective pharmacokinetics, nitrogen-containing bisphosphonates (N-BPs), currently used as clinical inhibitors of bone-resorption diseases, target osteoclast farnesyl pyrophosphate synthase (FPPS) and inhibit protein prenylation. FPPS, a key branchpoint of the mevalonate pathway, catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. To understand the molecular events involved in inhibition of FPPS by N-BPs, we used protein crystallography, enzyme kinetics, and isothermal titration calorimetry. We report here high-resolution x-ray structures of the human enzyme in complexes with risedronate and zoledronate, two of the leading N-BPs in clinical use. These agents bind to the dimethylallyl/geranyl pyrophosphate ligand pocket and induce a conformational change. The interactions of the N-BP cyclic nitrogen with Thr-201 and Lys-200 suggest that these inhibitors achieve potency by positioning their nitrogen in the proposed carbocation-binding site. Kinetic analyses reveal that inhibition is competitive with geranyl pyrophosphate and is of a slow, tight binding character, indicating that isomerization of an initial enzyme-inhibitor complex occurs with inhibitor binding. Isothermal titration calorimetry indicates that binding of N-BPs to the apoenzyme is entropy-driven, presumably through desolvation entropy effects. These experiments reveal the molecular binding characteristics of an important pharmacological target and provide a route for further optimization of these important drugs.

Animals↗

PubMed2006

Bone biology and the pathogenesis of osteoporosis.

Much is now known about skeletal biology and the changes that take place during diseases. Skeletal development is programmed by the sequential activation of specific genetic pathways that culminate in the production of the adult skeleton, which is light but strong. Systemic hormones including parathyroid hormone, vitamin D metabolites, and calcitonin regulate blood calcium levels and contribute to the overall calcium economy of the body. Many other hormones have subtle but important effects on skeletal behaviour and its modelling and remodelling activity. At a local level, the integration of cellular differentiation and function within the microenvironment of bone is under the influence of a large number of cytokines and growth factors. Osteoporosis is a very common disorder and is a result of perturbation in these regulatory mechanisms. Much has been learnt in recent years about the many pathogenic processes that contribute to bone loss and fragility. Several drug treatments are now available to prevent bone loss and reduce the incidence of fractures, and there are prospects for the development of further novel pharmacological interventions that may modify some of the pathogenic processes themselves. Among the newer pathways for pharmacological intervention, the calcium-sensing receptor and the receptor activator of nuclear factor kappa B ligand/receptor activator of nuclear factor kappa B/osteoprotegerin system involved in osteoblast-osteoclast interactions offer exciting opportunities.

Aging↗

PubMed2006

Bisphosphonates: from bench to bedside.

The discovery and development of the bisphosphonates (BPs) as a major class of drugs for the treatment of bone diseases has been a fascinating journey that is still not over. In clinical medicine, several BPs are established as the treatments of choice for various diseases of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. Bisphosphonates are chemically stable analogues of inorganic pyrophosphate, and are resistant to breakdown by enzymatic hydrolysis. Bisphosphonates inhibit bone resorption by being selectively taken up and adsorbed to mineral surfaces in bone, where they interfere with the action of the bone-resorbing osteoclasts. Bisphosphonates are internalized by osteoclasts and interfere with specific biochemical processes. Bisphosphonates can be classified into at least two groups with different molecular modes of action. The simpler non-nitrogen-containing bisphosphonates (such as clodronate and etidronate) can be metabolically incorporated into nonhydrolyzable analogues of adenosine triphosphate (ATP) that may inhibit ATP-dependent intracellular enzymes. The more potent, nitrogen-containing bisphosphonates (such as pamidronate, alendronate, risedronate, ibandronate, and zoledronate) are not metabolized in this way but can inhibit enzymes of the mevalonate pathway, thereby preventing the biosynthesis of isoprenoid compounds that are essential for the posttranslational modification of small GTP-binding proteins (which are also GTPases) such as rab, rho, and rac. The inhibition of protein prenylation and the disruption of the function of these key regulatory proteins explain the loss of osteoclast activity and induction of apoptosis. The key target for bisphosphonates is farnesyl pyrophosphate synthase (FPPS) within osteoclasts, and the recently elucidated crystal structure of this enzyme reveals how BPs bind to and inhibit at the active site via their critical N atoms. In conclusion, bisphosphonates are now established as an important class of drugs for the treatment of many bone diseases, and their mode of action is being unraveled. As a result their full therapeutic potential is gradually being realized.

Bone Density Conservation Agents↗

PubMed2005

Association of sporadic chondrocalcinosis with a -4-basepair G-to-A transition in the 5'-untranslated region of ANKH that promotes enhanced expression of ANKH protein and excess generation of extracellular inorganic pyrophosphate.

OBJECTIVE: Certain mutations in ANKH, which encodes a multiple-pass transmembrane protein that regulates inorganic pyrophosphate (PPi) transport, are linked to autosomal-dominant familial chondrocalcinosis. This study investigated the potential for ANKH sequence variants to promote sporadic chondrocalcinosis. METHODS: ANKH variants identified by genomic sequencing were screened for association with chondrocalcinosis in 128 patients with severe sporadic chondrocalcinosis or pseudogout and in ethnically matched healthy controls. The effects of specific variants on expression of common markers were evaluated by in vitro transcription/translation. The function of these variants was studied in transfected human immortalized CH-8 articular chondrocytes. RESULTS: Sporadic chondrocalcinosis was associated with a G-to-A transition in the ANKH 5'-untranslated region (5'-UTR) at 4 bp upstream of the start codon (in homozygotes of the minor allele, genotype relative risk 6.0, P = 0.0006; overall genotype association P = 0.02). This -4-bp transition, as well as 2 mutations previously linked with familial and sporadic chondrocalcinosis (+14 bp C-to-T and C-terminal GAG deletion, respectively), but not the French familial chondrocalcinosis kindred 143-bp T-to-C mutation, increased reticulocyte ANKH transcription/ANKH translation in vitro. Transfection of complementary DNA for both the wild-type ANKH and the -4-bp ANKH protein variant promoted increased extracellular PPi in CH-8 cells, but unexpectedly, these ANKH mutants had divergent effects on the expression of extracellular PPi and the chondrocyte hypertrophy marker, type X collagen. CONCLUSION: A subset of sporadic chondrocalcinosis appears to be heritable via a -4-bp G-to-A ANKH 5'-UTR transition that up-regulates expression of ANKH and extracellular PPi in chondrocyte cells. Distinct ANKH mutations associated with heritable chondrocalcinosis may promote disease by divergent effects on extracellular PPi and chondrocyte hypertrophy, which is likely to mediate differences in the clinical phenotypes and severity of the disease.

5' Untranslated Regions↗

PubMed2004

Ibandronate in osteoporosis: preclinical data and rationale for intermittent dosing.

Ibandronate is a highly potent, nitrogen-containing bisphosphonate. Unlike most other bisphosphonates, it is under clinical development for both oral and intravenous (i.v.) administration. Ibandronate can be used in convenient intermittent regimens that may optimize therapeutic outcome with enhanced compliance by patients. The preclinical pharmacokinetics (PK) and pharmacology of ibandronate have been extensively explored in a large preclinical development program involving various recommended animal models of human osteoporosis. These experimental studies of ibandronate indicate that the preclinical pharmacology and PK profile of ibandronate are broadly similar to those of other nitrogen-containing bisphosphonates. The efficacy of intermittent administration of subcutaneous (s.c.) and i.v. ibandronate has been demonstrated in four animal models (rat, dog, minipig, and monkey). Thus in rats, dogs, and monkeys with estrogen depletion, and in minipigs with glucocorticoid-induced bone loss, ibandronate administered s.c. or i.v. with extended intervals between doses reduces bone turnover, increases bone mineral density, and maintains bone quality in a dose-dependent manner. Furthermore, studies in rats and dogs comparing continuous and intermittent treatment schedules indicate similar efficacy when the same cumulative dose is applied over the duration of the study. These studies with ibandronate illustrate the concept that the total cumulative dose of bisphosphonate administered determines the response, independent of whether the dose is given daily or less frequently in a given time period. The efficacy of intermittent regimens has also been verified in models of secondary osteoporosis due to secondary hyperparathyroidism or immobilization (both in rats), or due to glucocorticoids in minipigs. Important factors for determining efficacy and the magnitude of response are the doses given, the length of the interval between doses, and the underlying bone turnover rate. The mechanisms underlying the remarkable efficacy of intermittent bisphosphonate dosing are not fully understood and further research is needed. Importantly, ibandronate is the only bisphosphonate so far proven to reduce the risk of vertebral fractures significantly with a between-dose interval >2 months, in a prospective clinical trial. Collectively, the preclinical studies on ibandronate have provided a sound basis for the design of the convenient regimens currently being examined in clinical trials.

Animals↗

PubMed2002

Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH.

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5p15. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

Amino Acid Sequence↗