Charles E. McKenna

The relationship between the chemistry and biological activity of the bisphosphonates

The ability of bisphosphonates ((HO)(2)P(O)CR(1)R(2)P(O)(OH)(2)) to inhibit bone resorption has been known since the 1960s, but it is only recently that a detailed molecular understanding of the relationship between chemical structures and biological activity has begun to emerge. The early development of chemistry in this area was largely empirical and based on modifying R(2) groups in a variety of ways. Apart from the general ability of bisphosphonates to chelate Ca(2+) and thus target the calcium phosphate mineral component of bone, attempts to refine clear structure-activity relationships had led to ambiguous or seemingly contradictory results. However, there was increasing evidence for cellular effects, and eventually the earliest bisphosphonate drugs, such as clodronate (R(1)=R(2)=Cl) and etidronate (R(1)=OH, R(2)=CH(3)), were shown to exert intracellular actions via the formation in vivo of drug derivatives of ATP. The observation that pamidronate, a bisphosphonate with R(1)=OH and R(2)=CH(2)CH(2)NH(2), exhibited higher potency than previously known bisphosphonate drugs represented the first step towards the later recognition of the critical importance of having nitrogen in the R(2) side chain. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates took place particularly in the 1980s, but still with an incomplete understanding of their structure-activity relationships. A major advance was the discovery that the anti-resorptive effects of the nitrogen-containing bisphosphonates (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their potency as inhibitors of the enzyme farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids utilized in sterol synthesis and for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of bisphosphonates have been synthesized and studied. Interest in expanding the structural scope of the bisphosphonate class has also motivated new approaches to the chemical synthesis of these compounds. Recent chemical innovations include the synthesis of fluorescently labeled bisphosphonates, which has enabled studies of the biodistribution of these drugs. As a class, bisphosphonates share common properties. However, as with other classes of drugs, there are chemical, biochemical, and pharmacological differences among the individual compounds. Differences in mineral binding affinities among bisphosphonates influence their differential distribution within bone, their biological potency, and their duration of action. The overall pharmacological effects of bisphosphonates on bone, therefore, appear to depend upon these two key properties of affinity for bone mineral and inhibitory effects on osteoclasts. The relative contributions of these properties differ among individual bisphosphonates and help determine their clinical behavior and effectiveness.

Fluorescent risedronate analogues reveal bisphosphonate uptake by bone marrow monocytes and localization around osteocytes in vivo.

Bisphosphonates are effective antiresorptive agents owing to their bone‐targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non‐osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein‐labeled risedronate (FAM‐RIS) and Alexa Fluor 647–labeled risedronate (AF647‐RIS), were used to address this question. Twenty‐four hours after injection into 3‐month‐old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM‐RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14high bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14+ cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone‐resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo.

A film bulk acoustic resonator in liquid environments

A highly sensitive film bulk acoustic resonator (FBAR) mass sensor in liquid environments is described in this paper. A transmission line model is used to theoretically predict the dependence of the FBARs resonant frequency on added mass. FBAR performance in a liquid environment is experimentally characterized for the first time and the effects of the liquid nature and conductivity on the FBAR series and parallel resonant frequencies are investigated. A TiO2-coated FBAR is developed for sensitive mass sensing of metal ions in a liquid environment.

Bromodomains: Structure, function and pharmacology of inhibition

Bromodomains are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation. The human proteome comprises 46 bromodomain-containing proteins with a total of 61 bromodomains, which, despite highly conserved structural features, recognize a wide array of natural peptide ligands. Over the past five years, bromodomains have attracted great interest as promising new epigenetic targets for diverse human diseases, including inflammation, cancer, and cardiovascular disease. The demonstration in 2010 that two small molecule compounds, JQ1 and I-BET762, potently inhibit proteins of the bromodomain and extra-terminal (BET) family with translational potential for cancer and inflammatory disease sparked intense efforts in academia and pharmaceutical industry to develop novel bromodomain antagonists for therapeutic applications. Several BET inhibitors are already in clinical trials for hematological malignancies, solid tumors and cardiovascular disease. Currently, the field faces the challenge of single-target selectivity, especially within the BET family, and of overcoming problems related to the development of drug resistance. At the same time, new trends in bromodomain inhibitor research are emerging, including an increased interest in non-BET bromodomains and a focus on drug synergy with established antitumor agents to improve chemotherapeutic efficacy. This review presents an updated view of the structure and function of bromodomains, traces the development of bromodomain inhibitors and their potential therapeutic applications, and surveys the current challenges and future directions of this vibrant new field in drug discovery.

RAB26 and RAB3D are direct transcriptional targets of MIST1 that regulate exocrine granule maturation

ABSTRACT Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. MIST1, an evolutionarily conserved transcription factor, is required for the formation of large, specialized secretory vesicles in gastric zymogenic (chief) cells (ZCs) as they differentiate from their mucous neck cell progenitors. Here, we show that MIST1 binds to highly conserved CATATG E-boxes to directly activate transcription of 6 genes, including those encoding the small GTPases RAB26 and RAB3D. We next show that RAB26 and RAB3D expression is significantly downregulated in Mist1−/− ZCs, suggesting that MIST1 establishes large secretory granules by inducing RAB transcription. To test this hypothesis, we transfected human gastric cancer cell lines stably expressing MIST1 with red fluorescent protein (RFP)-tagged pepsinogen C, a key secretory product of ZCs. Those cells upregulate expression of RAB26 and RAB3D to form large secretory granules, whereas control, non-MIST1-expressing cells do not. Moreover, granule formation in MIST1-expressing cells requires RAB activity because treatment with a RAB prenylation inhibitor and transfection of dominant negative RAB26 abrogate granule formation. Together, our data establish the molecular process by which a transcription factor can directly induce fundamental cellular architecture changes by increasing transcription of specific cellular effectors that act to organize a unique subcellular compartment.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower‐affinity analogues deoxy‐risedronate and 3‐PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower‐affinity compounds showed preferential binding to resorption lacunae, whereas the highest‐affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower‐affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high‐ and low‐affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that “recycling” had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton.

Real-Time Intravital Imaging Establishes Tumor-Associated Macrophages as the Extraskeletal Target of Bisphosphonate Action in Cancer

UNLABELLED Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their antiresorptive effects on the skeleton. However, because bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their antitumor action, particularly on cells outside of bone, remain unknown. Here, we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumors, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumor-associated macrophages (TAM), but not tumor cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumor of a patient and showed the resected tumor to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumors outside the skeleton and that their antitumor activity is likely to be mediated via TAMs. SIGNIFICANCE Bisphosphonates are assumed to act solely in bone. However, mouse models and clinical trials show that they have surprising antitumor effects outside bone. We provide unequivocal evidence that bisphosphonates target TAMs, but not tumor cells, to exert their extraskeletal effects, offering a rationale for use in patients with early disease.

Identification of a novel BET bromodomain inhibitor‐sensitive, gene regulatory circuit that controls Rituximab response and tumour growth in aggressive lymphoid cancers

Immuno‐chemotherapy elicit high response rates in B‐cell non‐Hodgkin lymphoma but heterogeneity in response duration is observed, with some patients achieving cure and others showing refractory disease or relapse. Using a transcriptome‐powered targeted proteomics screen, we discovered a gene regulatory circuit involving the nuclear factor CYCLON which characterizes aggressive disease and resistance to the anti‐CD20 monoclonal antibody, Rituximab, in high‐risk B‐cell lymphoma. CYCLON knockdown was found to inhibit the aggressivity of MYC‐overexpressing tumours in mice and to modulate gene expression programs of biological relevance to lymphoma. Furthermore, CYCLON knockdown increased the sensitivity of human lymphoma B cells to Rituximab in vitro and in vivo. Strikingly, this effect could be mimicked by in vitro treatment of lymphoma B cells with a small molecule inhibitor for BET bromodomain proteins (JQ1). In summary, this work has identified CYCLON as a new MYC cooperating factor that autonomously drives aggressive tumour growth and Rituximab resistance in lymphoma. This resistance mechanism is amenable to next‐generation epigenetic therapy by BET bromodomain inhibition, thereby providing a new combination therapy rationale for high‐risk lymphoma.

A method for preparing analytically pure sodium dithionite. Dithionite quality and observed nitrogenase-specific activities

Sodium dithionite (Na2S2O4) is widely used as a reductant in biochemical studies, but has not been available in its pure form. A convenient, detailed procedure is given for the recrystallization of commercial dithionite from 0.1 M NaOH-methanol under anaerobic conditions. Twice-recrystallized dithionite had a purity of 99 +/- 1% by UV spectroscopy (A315) and elemental analysis. The influence of dithionite quality on the apparent reduction activities of the nitrogenase components (Av1 and Av2) from Azotobacter vinelandii was investigated.

Synthesis, Chiral High Performance Liquid Chromatographic Resolution and Enantiospecific Activity of a Potent New Geranylgeranyl Transferase Inhibitor, 2-Hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic Acid

3-(3-Pyridyl)-2-hydroxy-2-phosphonopropanoic acid (3-PEHPC, 1) is a phosphonocarboxylate (PC) analogue of 2-(3-pyridyl)-1-hydroxyethylidenebis(phosphonic acid) (risedronic acid, 2), an osteoporosis drug that decreases bone resorption by inhibiting farnesyl pyrophosphate synthase (FPPS) in osteoclasts, preventing protein prenylation. 1 has lower bone affinity than 2 and weakly inhibits Rab geranylgeranyl transferase (RGGT), selectively preventing prenylation of Rab GTPases. We report here the synthesis and biological studies of 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC, 3), the PC analogue of minodronic acid 4. Like 1, 3 selectively inhibited Rab11 vs. Rap 1A prenylation in J774 cells, and decreased cell viability, but was 33-60x more active in these assays. After resolving 3 by chiral HPLC (>98% ee), we found that (+)-3-E1 was much more potent than (-)-3-E2 in an isolated RGGT inhibition assay, approximately 17x more potent (LED 3 microM) than (-)-3-E2 in inhibiting Rab prenylation in J774 cells and >26x more active in the cell viability assay. The enantiomers of 1 exhibited a 4-fold or smaller potency difference in the RGGT and prenylation inhibition assays.