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The results of a solid-state [super 13]C, [super 15]N, and [super 31]P magic-angle sample spinning (MAS) NMR and quantum chemical investigation of several bisphosphonates, both as pure compounds and when bound to farnesyl diphosphate synthase (FPPS) is reported. Bisphosphonates are a class of molecules in widespread use in treating bone resorption diseases and are also of interest as immunomodulators and anti-infectives.

Research has been conducted on the base methylation important for the DNA biological functions. The changes in the DNA local dynamics upon substitutions of a methylated cytosine analogue for cytosine have been investigated via the use of the solid-state deuterium NMR and the results are discussed.

Research has been conducted on the perturbations of the EcoRI restriction binding site internal dynamics. The use of solid-state deuterium magnetic resonance imaging in investigating these perturbations is described.

A deuterium nuclear magnetic resonance study was conducted on the mobility at the C9pG10 step in the in the DNA dodecamer [d(CGCGAATTCGCG)]2 to examine the internal, localized dynamics of DNA. Results show that the amplitude and rate of the uniform rotation of the dC9-labeled oligomer are sensitive to hydration level. The Zeeman relaxation times for the furanose ring and backbone deuterons are the shortest for any deuteron in the DNA dodecamer at hydration levels of about 10-12 waters per nucleotide.

The dynamics of the phosphodiester backbone in the [[5'-GCGC-3'].sub.2] moiety of the DNA oligomer are analyzed by using deuterium solid-state NMR (SSNMR). The NMR data has shown that the [[5'-GCGC-3'].sub.2] moiety is dynamic and the change of orientation of this moiety in DNA upon methylation has made the molecule less amenable to binding to the HhaI endonuclease.

A deuterium solid-state NMR (SSNMR) was used to study the dynamics of the furanose rings in the GCGC moiety of the DNA oligomer [[d([G.sub.1][A.sub.2][T.sub.3][A.sub.4][G.sub.5][C.sub.6][G.sub.7][C.sub.8][T.sub.9][A.sub.10][T.sub.11][C.sub.12])].sub.2]. The NMR data results shed light onto the dynamic property of 5'-GCGC-3' with the largest-amplitude motions occurring nearest the methylation site and its structural rearrangements when the DNA binds to the Hhal methyltransferase.

Solid state and solution NMR are used to investigate internal motions at six positions near the HhaI methyltransferase enzyme recognition site for the [[5'-(dGATAGCGCTATC)-3'].sub.2] DNA dodecamer. The results have shown that the HhaI DNA methylation target site possesses an intrinsically dynamic structure and the measurements of dynamics for many nucleotides in the vicinity of the methylation site have identified two residues that experience significant mobility in the absence of the methylase.

Protein-DNA interactions play an important role in numerous biological functions within the living cell. In many of these interactions, the DNA helix is significantly distorted upon protein-DNA complex formation. The Hha I restriction-modification system is one such system, where the methylation target is flipped out of the helix when bound to the methyltransferase. However, the base flipping mechanism is not well understood. The dynamics of the binding site of the Hha I methyltransferase and endonuclease (underlined) within the DNA oligomer [d(G 1 A 2 T 3 A 4G5C6G7C8 T 9 A 10 T 11 C 12 )] 2 are studied using deuterium solid-state NMR (SSNMR). SSNMR spectra obtained from DNAs deuterated on the base of nucleotides within and flanking the [5'-GCGC-3'] 2 sequence indicate that all of these positions are structurally flexible. Previously, conformational flexibility within the phosphodiester backbone and furanose ring within the target sequence has been observed and hypothesized to play a role in the distortion mechanism. However, whether that distortion was occurring through an active or passive mechanism remained unclear. These NMR data demonstrate that although the [5'-GCGC-3'] 2 sequence is dynamic, the target cytosine is not passively flipping out of the double-helix on the millisecond-picosecond time scale. Additionally, although previous studies have shown that both the furanose ring and phosphodiester backbone experience a change in dynamics upon methylation, which may play a role in recognition and cleavage by the endonuclease, our observations here indicate that methylation has no effect on the dynamics of the base itself.

Bisphosphonates are a class of molecules in widespread use in treating bone resorption diseases and are also of interest as immunomodulators and anti-infectives. They function by inhibiting the enzyme farnesyl diphosphate synthase (FPPS), but the details of how these molecules bind are not fully understood. Here, we report the results of a solid-state (13)C, (15)N, and (31)P magic-angle sample spinning (MAS) NMR and quantum chemical investigation of several bisphosphonates, both as pure compounds and when bound to FPPS, to provide information about side-chain and phosphonate backbone protonation states when bound to the enzyme. We then used computational docking methods (with the charges assigned by NMR) to predict how several bisphosphonates bind to FPPS. Finally, we used X-ray crystallography to determine the structures of two potent bisphosphonate inhibitors, finding good agreement with the computational results, opening up the possibility of using the combination of NMR, quantum chemistry and molecular docking to facilitate the design of other, novel prenytransferase inhibitors.

The effects of a series of 102 bisphosphonates on the inhibition of growth of Entamoeba histolytica and Plasmodium falciparum in vitro have been determined, and selected compounds were further investigated for their in vivo activity. Forty-seven compounds tested were active (IC(50) < 200 microM) versus E. histolytica growth in vitro. The most active compounds (IC(50) approximately 4-9 microM) were nitrogen-containing bisphosphonates with relatively large aromatic side chains. Simple n-alkyl-1-hydroxy-1,1-bisphosphonates, known inhibitors of the enzyme farnesylpyrophosphate (FPP) synthase, were also active, with optimal activity being found with C9-C10 side chains. However, numerous other nitrogen-containing bisphosphonates known to be potent FPP synthase inhibitors, such as risedronate or pamidronate, had little or no activity. Several pyridine-derived bisphosphonates were quite active (IC(50) approximately 10-20 microM), and this activity was shown to correlate with the basicity of the aromatic group, with activity decreasing with increasing pK(a) values. The activities of all compounds were tested versus a human nasopharyngeal carcinoma (KB) cell line to enable an estimate of the therapeutic index (TI). Five bisphosphonates were selected and then screened for their ability to delay the development of amebic liver abscess formation in an E. histolytica infected hamster model. Two compounds were found to decrease liver abscess formation at 10 mg/kg ip with little or no effect on normal liver mass. With P. falciparum, 35 compounds had IC(50) values <200 microM in an in vitro assay. The most active compounds were also simple n-alkyl-1-hydroxy-1,1-bisphosphonates, having IC(50) values around 1 microM. Five compounds were again selected for in vivo investigation in a Plasmodium berghei ANKA BALB/c mouse suppressive test. The most active compound, a C9 n-alkyl side chain containing bisphosphonate, caused an 80% reduction in parasitemia with no overt toxicity. Taken together, these results show that bisphosphonates appear to be useful lead compounds for the development of novel antiamebic and antimalarial drugs.