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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.

Differences in the binding affinities of bisphosphonates for bone mineral have been proposed to determine their localizations and duration of action within bone. The main objective of this study was to test the hypothesis that mineral binding affinity affects bisphosphonate distribution at the basic multicellular unit (BMU) level within both cortical and cancellous bone. To accomplish this objective, skeletally mature female rabbits (n = 8) were injected simultaneously with both low- and high-affinity bisphosphonate analogs bound to different fluorophores. Skeletal distribution was assessed in the rib, tibia, and vertebra using confocal microscopy. The staining intensity ratio between osteocytes contained within the cement line of newly formed rib osteons or within the reversal line of hemiosteons in vertebral trabeculae compared to osteocytes outside the cement/reversal line was greater for the high-affinity compared to the low-affinity compound. This indicates that the low-affinity compound distributes more equally across the cement/reversal line compared to a high-affinity compound, which concentrates mostly near surfaces. These data, from an animal model that undergoes intracortical remodeling similar to humans, demonstrate that the affinity of bisphosphonates for the bone determines the reach of the drugs in both cortical and cancellous bone.

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. © 2010 American Society for Bone and Mineral Research

Local delivery of bisphosphonates results in superior localization of these compounds for the treatment of cochlear otosclerosis, without ototoxicity.Otosclerosis is a common disorder of abnormal bone remodeling within the human otic capsule. It is a frequent cause of conductive hearing loss from stapes fixation. Large lesions that penetrate the cochlear endosteum and injure the spiral ligament result in sensorineural hearing loss. Nitrogen-containing bisphosphonates (e.g., zoledronate) are potent inhibitors of bone remodeling with proven efficacy in the treatment of metabolic bone diseases, including otosclerosis. Local delivery to the cochlea may allow for improved drug targeting, higher local concentrations, and the avoidance of systemic complications. In this study, we use a fluorescently labeled bisphosphonate compound (6-FAM-ZOL) to determine drug localization and concentration within the otic capsule. Various methods for delivery are compared. Ototoxicity is evaluated by auditory brainstem responses and distortion product otoacoustic emissions.6-FAM-ZOL was administered to guinea pigs via intraperitoneal injection, placement of alginate beads onto the round window membrane, or microfluidic pump infusion via a cochleostomy. Hearing was evaluated. Specimens were embedded into resin blocks, ground to a mid-modiolar section, and quantitatively imaged using fluorescence microscopy.There was a dose-dependent increase in fluorescent signal after systemic 6-FAM-ZOL treatment. Local delivery via the round window membrane or a cochleostomy increased delivery efficiency. No significant ototoxicity was observed after either systemic or local 6-FAM-ZOL delivery.These findings establish important preclinical parameters for the treatment of cochlear otosclerosis in humans.

Novel α,β-CH2 and β,γ-NH (1a) or α,β-NH and β,γ-CH2 (1b) “Met-Im” dTTPs were synthesized via monodemethylation of triethyl-dimethyl phosphorimido-bisphosphonate synthons (4a, 4b), formed via a base-induced [1,3]-rearrangement of precursors (3a, 3b) in a reaction with dimethyl or diethyl phosphochloridate. Anomerization during final bromotrimethylsilane (BTMS) deprotection after Mitsunobu conjugation with dT was avoided by microwave conditions. 1a was 9-fold more potent in inhibiting DNA polymerase β, attributed to an NH-group interaction with R183 in the active site.

Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. Here we investigate a chromatin-interacting module, the bromodomain (BD) from the BET family of proteins, as a potential antifungal target in Candida albicans, a major human fungal pathogen. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice. We report small-molecule compounds that inhibit C. albicans Bdf1 with high selectivity over human BDs. Crystal structures of the Bdf1 BDs reveal binding modes for these inhibitors that are sterically incompatible with the human BET-binding pockets. Furthermore, we report a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 BD-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal therapeutic strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.

DNA polymerase (pol) β is a member of the X-family of DNA polymerases and plays an important role in base excision repair that cleanses the genome of simple base lesions.[1] It has been the subject of extensive studies examining its key roles in repair[2] and cancer.[3] In an effort to identify features of the pol β active site that modulate nucleotide binding, we have made modifications in the triphosphate moiety and studied the effect on enzyme binding, specificity, and chemistry.[4] Replacement of the Pα-O-Pβ bridging oxygen with a carbon atom prevents catalysis,[4b] whereas modification of the Pβ-O-Pγ bridging oxygen alters leaving group properties.[4a] The introduction of these substituents may also enable entirely new bonding (or repulsive) active site interactions, not present with the natural nucleoside triphosphate, and thus could inform mechanistic insight as well as inhibitor design seeking to exploit pol β as a drug target.[4a] Previously we studied several α,β-methylene-dNTP analogues as probes for the capture of pre-insertion substrate complexes with pol β and found that α,β-CF2-analogues have an apparent equilibrium dissociation constant (Kd) that is two orders of magnitude greater than the corresponding α,β-CH2-analogues and the β,γ-dGTP-CF2-analogue.[4a, 4b] To further explore the molecular basis of pol β nucleotide binding, the dimethylated (α,β-C(Me)2-, (5)) and the monofluorinated (R/S)-(α,β-CHF-, (6a/b)) dATP analogues have been synthesized and their Kd values determined. In addition, the X-ray crystallographic structures of ternary substrate complexes of pol β and DNA primer template formed from α,β-CF2-dATP[4b] (7) and 6a/b have been solved. Interestingly, only one diastereomer of 6 is found in the active site (Figure 1). Figure 1 X-ray crystal structure of the DNA pol β active site containing (S)-α,β-CHF-dATP. The solvent-excluded pol β active site surface is colored by atom (carbon, gray; nitrogen, light blue; oxygen, pink; magnesium, dark blue ... 5 and 6a/b were prepared by reaction of 2′-deoxyadenosine 5′-tosylate with the tris(tetrabutylammonium) salt of the corresponding bisphosphonic acid followed by enzymatic phosphorylation of the resulting dA 5′-bisphosphonate[4b] (Scheme 1). In a previously reported synthesis of 6a/b in which (R/S)-α,β-CHF-dADP (4a/b) was phosphorylated with p-nitrobenzyl phosphormorpholidate,[5] an approximately 1:1 mixture of α,β-CHF-dATP diastereomers was observed in the 19F NMR spectrum. Similarly, phosphorylation of 4a/b using nucleoside diphosphate kinase and a catalytic amount of ATP generates both diastereomers as observed in the 19F and 31P (Pα) NMR spectra. Although precise quantification is not possible due to signal overlap, by 19F NMR analysis the isomers are in roughly similar abundance, indicating that the enzymatic phosphorylation did not significantly enrich a particular isomer. Scheme 1 Synthesis of α,β-CXY-dATP analogues. As expected, 5 and 6a/b are not pol β substrates (SI, Figure S25) and inhibit gap-filling DNA synthesis by pol β in a concentration dependent manner (SI, Figures S26 and S28). The apparent equilibrium binding constant for 6a/b is approximately 10 μM, similar to that of the natural substrate and significantly lower than that of the α,β-CF2-dATP analogue (Figure 2).[4b] The α,β-CH2-dATP analogue binds with about a 10-fold higher affinity[4b, 6] than 6a/b consistent with a correlation between the basicity of the phosphonate moiety and the strength of inhibitor binding.[4b] However, α,β-C(Me)2-dATP (5) which has two electron-donating substituents on the α,β-methylene, has a Kd of approximately 1100 μM and does not conform to this trend. Figure 2 DNA pol β dissociation constants of dATP and α,β-methylene analogues. The data for dATP, α,β-CH2-dATP, and α,β-CF2-dATP were previously published.[4b, 9] The Ki (i.e., Kd) values were determined ... The crystallographic structures of ternary complexes of pol β with incoming α,β-CF2-dATP (7) (PDB ID 3TFR) or α,β-CHF-dATP (6) (PDB ID 3TFS) opposite a templating thymidine, resolved at 2.00 A (SI Table S1) were next determined. All corresponding atoms in the active site of these structures superimpose well with previously determined ternary complex structures of pol β where the reaction was trapped by deletion of the nucleophilic 3′-OH on the primer terminus or by using a nitrogen in place of the α,β-bridging oxygen (Figure 3A).[8] Thus, α,β-CHF-dATP and 7 are well accommodated in the polymerase active site as has been shown previously for α,β-CF2-dTTP.[4b] Figure 3 A) The ternary complex crystallographic structures of pol β with an incoming α,β-CF2-dATP (7) (gray carbons) and α,β-NH-dUTP (light blue carbons; PDB ID 2FMS)[8] were superimposed using all 326 Cα (rmsd ... Molecular docking of 5 and 6 with pol β-DNA binary complex using Autodock Vina[7] suggests that the unexpectedly low binding affinity of 5 can be attributed to an unfavorable steric interaction, specifically a clash of the methyl groups of 5 with a structural water bound to Asp276 (PDB ID 3TFS water 3). When docked in the absence of this structural water, the lowest energy conformations for both 5 and 6 overlap well with the coordinates of the 6 found in the crystal structure, whose conformation is congruent with those of DNA pol β-bound α,β-CF2-dTTP[4b] and α,β-NH-dUTP.[8] However, when docking using a macromolecule file prepared to include the water bound to Asp276, 5 is unable to achieve the experimentally observed conformation while the geometry of 6 is unperturbed (SI, Figure S27). Incubation of crystals of a binary DNA complex of pol β with the α,β-CHF-dATP diastereomer mixture (6a/b) resulted in a complex in which only the (S)-CHF-stereoisomer (6b) could be found by X-ray analysis (Figures 1 and ​and3B).3B). Modeling (R)-α,β-CFH-dATP (6a) into the electron density failed to account for the observed density (Figure 3C). The close overlap of the CF2- and the (S)-CHF- conformations indicates that exclusion of 6a analogue is not the result of an unattainable binding conformation or destabilizing steric interaction (Figure 3B). As we reasoned previously,[4d, 4e] stabilization of as little as ~1 kcal/mol could be sufficient to generate the selectivity observed in the crystal structures. In an earlier study, we reported a series of stereospecifically formed pol β-DNA-dGTP ternary complexes that provided evidence for a stabilizing polar interaction between the active site Arg183 and the chiral phosphonate C-F in CHF, CMeF, and CClF dGTP-β,γ analogues.[4d, 4e] Unlike the β,γ-CXF-dGTP-DNA-pol β ternary complexes, in this structure no direct stabilizing interactions with active site residues can be identified. The structure does, however, reveal that the (S)-isomer fluorine is within 2.8 A (B-factor: 15.4 A2) of an oxygen in a structural water molecule, present in both structures (for CF2 distance = 2.9 A, B-factor: 15.1 A2), that is within hydrogen bonding distance of several key atoms (a carboxylate anion oxygen and carboxyamide nitrogen of Asp276, phosphoryl oxygen of Pβ; Figure 1). Observing no similar interactions with the pro-(R)-fluorine in the CF2-structure, one might consider a weak interaction of the (S)-fluorine in 6b with structural water as the source of its stabilization in the complex relative to 6a, however other factors might be invoked[10] and further work will be necessary to clarify the origin of the observed stereospecificity. In summary, our results show the influence of a structural water bound to Asp276 on the nucleotide binding properties of DNA pol β. Kd determinations for a series of α,β-bridging substituted dATP analogues display a correlation with the basicity of the bisphosphonate moiety and the binding affinity to pol β. This trend, however, does not extend to the α,β-C(Me)2-dATP analogue which displays a larger than expected Kd attributed to an unfavorable steric interaction of the methyl substituents with the structural water bound to Asp276. The X-ray crystal structure of α,β-CHF-dATP in complex with pol β and DNA contains only one diastereomer despite the presence of both diastereomers in the crystallization mixture. Comparison of this structure with that of α,β-CF2-dATP raises the possibility of a non-covalent weak interaction between the ligand and the active site structural water proximal to Asp276. Our structural observations may be worth taking into account in PDB studies[11] searching for fluorine/protein interactions.

Progress in the synthesis of novel fluorescent conjugates of N-heterocyclic bisphosphonate drugs and related analogues, together with some recent applications of these compounds as imaging probes, are briefly discussed.

Rab geranylgeranyl transferase (RGGT) catalyzes the posttranslational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki. (+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and RabCXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases. publishersversion published

β,γ-Fluoromethylene analogues of nucleotides are generally considered to be useful mimics of the natural substrates for DNA polymerases, but direct structural evidence defining their active site interactions has not been available. In addition, the effect of introducing a new chiral center (the CHF carbon) has been unexplored. We report here structural studies of the diastereomeric β,γ-CHF analogues (R, 3; S, 4) of dGTP interacting with the active site of DNA pol β, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. The conjugation of dGMP 5‘-morpholidate with a tetrabutylammonium salt of (fluoromethylene)bisphosphonic acid (6b, prepared like its difluoro analogue 7b via fluorination of tetraisopropyl methylenebisphosphonate carbanion with Selectfluor) gives a 1:1 mixture of 3 and 4 (by 19F NMR, pH 10). The β,γ-CF2 (2) and β,γ-CH2 (1) dGTP analogues were also synthesized. Crystallization from a solution containing 3 + 4 together with a preformed DNA pol β complex o...