Identification and specificity profiling of protein prenyltransferase inhibitors using new fluorescent phosphoisoprenoids

Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.     

Ruthenium complexes with the stanna-closo-dodecaborate ligand: coexistence of eta1(Sn) and eta3(B-H) coordination

Four stanna-closo-dodecaborate complexes of ruthenium have been prepared and characterized by multinuclear NMR studies in solution and in the solid state. The solid-state structures of the dimeric zwitterions [[Ru(dppb)(SnB11H11)]2] (2) (dppb = bis(diphenylphosphino)butane), [[Ru(PPh3)2(SnB11H11)]2] (3), and the dianionic ruthenium complex [Bu3MeN]2[Ru(dppb)[2,7,8-(mu-H)3-exo-SnB11H11](SnB11H11)] (4) were determined by X-ray crystal structure analysis; they establish an unprecedented structural motif in the chemistry of heteroboranes and transition-metal fragments with the stanna-closo-dodecaborate moiety as a two-faced ligand that exhibits eta1(Sn) as well as eta3(B-H) coordination. The eta3-coordinated stannaborate in 4 and in the isostructural compound [Bu3MeN]2[Ru(PPh3)2[2,7,8-(mu-H)3-exo-SnB11H11](SnB11H11)] (5) shows fluxional behavior, which was studied in detail by using 31P[1H] EXSY and DNMR experiments. The activation parameters for the dynamic process of 5 are given.     

Nickel quantification in serum by a validated sector-field inductively coupled plasma mass spectrometry method: Assessment of tentative reference values for an Italian population

The daily exposure to Ni from food, industrial processes, jewellery and coins makes the determination of Ni in human serum an important way to monitor the health status in non-occupationally exposed subjects. To this end, a method based on sector-field inductively coupled plasma mass spectrometry was developed and validated. The limits of detection (LoD) and quantification (LoQ), sensitivity, linearity range, trueness, repeatability, within-laboratory reproducibility and robustness were the considered issues of the validation process. The uncertainty associated with the measurements was also calculated, according to the Eurachem/Citac Guide. The method LoD and LoQ were 0.03 and 0.09 ng mL(-1), linearity was over two order of magnitude, trueness was -3.57%, and the repeatability and reproducibility showed relative standard deviations equal to 4.56% and 6.52%, respectively. The relative expanded uncertainty was 21.8% at the Ni levels found in the general population. The tentative reference value for serum Ni was 0.466 +/- 0.160 ng mL(-1) with a related interval between 0.226 and 1.026 ng mL(-1).     

Design, synthesis, and biological evaluation of somatostatin-based radiopeptides

The prototypes for tumor targeting with radiolabeled peptides are derivatives of somatostatin. Usually, they primarily have high affinity for somatostatin receptor subtype 2 (sst2), and they have moderate affinity for sst5. We aimed at developing analogs that recognize different somatostatin receptor subtypes for internal radiotherapy in order to extend the present range of accessible tumors. We synthesized DOTA-octapeptides based on octreotide by replacing Phe3 mainly with unnatural amino acids. The affinity profile was determined by using cell lines transfected with sst1-5. Internalization was determined by using AR42J, HEK-sst3, and HEK-sst5 cell lines, and biodistribution was studied in rat tumor models. Two of the derivatives thus obtained showed an improved binding affinity profile, enhanced internalization into cells expressing sst2 and sst3, respectively, and better tumor:kidney ratios in animals.     

Robust packing in cyclopalladated primary amines: isomorphous crystal structures of four complexes with varying substitution patterns

The crystal structures of (SP‐4‐4)‐[rac‐2‐(1‐aminoethyl)phenyl‐κ²C¹,N]chlorido(pyridine‐κN)palladium(II), [Pd(C₈H₁₀N)Cl(C₅H₅N)], (I), (SP‐4‐4)‐[rac‐2‐(1‐aminoethyl)phenyl‐κ²C¹,N]bromido(pyridine‐κN)palladium(II), [PdBr(C₈H₁₀N)(C₅H₅N)], (II), (SP‐4‐4)‐[rac‐2‐(1‐aminoethyl)‐5‐bromophenyl‐κ²C¹,N]bromido(4‐methylpyridine‐κN)palladium(II), [PdBr(C₈H₉BrN)(C₆H₇N)], (III), and (SP‐4‐4)‐[rac‐2‐(1‐aminoethyl)‐5‐bromophenyl‐κ²C¹,N]iodido(4‐methylpyridine‐κN)palladium(II), [Pd(C₈H₉BrN)I(C₆H₇N)], (IV), are reported. The latter is the first iodide complex in this class of compounds. All four complexes crystallize in the same space group, viz.I4₁/a, with very similar lattice parameters a and more flexible lattice parameters c. Their packing corresponds to that of their enantiomerically pure congeners, which crystallize in the t2 subgroup I4₁.     

Spatial perception and adaptive sonar behavior

Bat echolocation is a dynamic behavior that allows for real-time adaptations in the timing and spectro-temporal design of sonar signals in response to a particular task and environment. To enable detailed, quantitative analyses of adaptive sonar behavior, echolocation call design was investigated in big brown bats, trained to rest on a stationary platform and track a tethered mealworm that approached from a starting distance of about 170 cm in the presence of a stationary sonar distracter. The distracter was presented at different angular offsets and distances from the bat. The results of this study show that the distance and the angular offset of the distracter influence sonar vocalization parameters of the big brown bat, Eptesicus fuscus. Specifically, the bat adjusted its call duration to the closer of two objects, distracter or insect target, and the magnitude of the adjustment depended on the angular offset of the distracter. In contrast, the bat consistently adjusted its call rate to the distance of the insect, even when this target was positioned behind the distracter. The results hold implications for understanding spatial information processing and perception by echolocation.     

Enantiospecific Synthesis of ortho‐Substituted 1,1‐Diarylalkanes by a 1,2‐Metalate Rearrangement/anti‐SN2′ Elimination/Rearomatizing Allylic Suzuki–Miyaura Reaction Sequence

The one‐pot sequential coupling of benzylamines, boronic esters, and aryl iodides has been investigated. In the presence of an N‐activator, the boronate complex formed from an ortho‐lithiated benzylamine and a boronic ester undergoes stereospecific 1,2‐metalate rearrangement/anti‐S_N2′ elimination to form a dearomatized tertiary boronic ester. Treatment with an aryl iodide under palladium catalysis leads to rearomatizing γ‐selective allylic Suzuki–Miyaura cross‐coupling to generate 1,1‐diarylalkanes. When enantioenriched α‐substituted benzylamines are employed, the corresponding 1,1‐diarylalkanes are formed with high stereospecificity.     

Duplex‐Guided Refolding into Novel G‐Quadruplex (3+1) Hybrid Conformations

The oligomer d(GCGTG₃TCAG₃TG₃TG₃ACGC) with short complementary flanking sequences at the 5′‐ and 3′‐ends was shown to fold into three different DNA G‐quadruplex species. In contrast, a corresponding oligomer that lacks base complementarity between the two overhang sequences folds into a single parallel G‐quadruplex. The three coexisting quadruplex structures were unambiguously identified and structurally characterized through detailed spectral comparisons with well‐defined G‐quadruplexes formed upon the deliberate incorporation of syn‐favoring 8‐bromoguanosine analogues into specific positions of the G‐core. Two (3+1) hybrid structures coexist with the parallel fold and feature a novel lateral–propeller–propeller loop architecture that has not yet been confirmed experimentally. Both hybrid quadruplexes adopt the same topology and only differ in their pattern of anti→syn transitions and tetrad stackings.     

Synthesis and endosomolytic properties of poly(amidoamine) block copolymers

The poly(amidoamine)s (PAAs) ISA 1 and ISA 23 display pH-dependent conformational change and pH-dependent membrane perturbation. These properties confer potential for use as endosomolytic polymers for intracytoplasmic delivery of toxins and genes. Both polymers are relatively non-toxic, and moreover ISA 23 has the beneficial property in vivo, of being non hepatotropic when administered intravenously. Although ISA 23 and ISA 1 demonstrate ability to transfect cells, ISA 1 is also able to promote intracellular delivery of non-permeant toxins. The aim of this study was to synthesise random and block copolymers of ISA 1 and ISA 23 and investigate whether these second generation hybrids would allow optimisation of PAA biological characteristics. Random and block copolymers of ISA 1 and ISA 23 were synthesised by hydrogen transfer polyaddition to generate a library of PAAs with an ISA 23:ISA 1 molar ratios of 2:1 to 4:1. The resultant polymers have a pI slightly below 7.4 and a M(w) of 19,900-49,000 g/mol and a M(n) of 13,100-24,100 g/mol. Whereas none of the random or block copolymers were haemolytic at pH 7.4 all demonstrated pH-dependent membrane activity. At pH 5.5 they caused 50-60% haemoglobin (Hb) release over 1 h. This was slightly less than that seen for ISA 23 (80% Hb release). None of the copolymers were cytotoxic against B16F10 cells during a 72 h incubation (IC(50) > 2 mg/ml; MTT assay). The ability of the random and block copolymer PAAs to deliver the toxin gelonin was also examined, but only ISA 1 and the block copolymer B2 (ISA 23:ISA 1 at a 2:1 molar ratio) were able to promote intracellular delivery, as measured by cytotoxic activity. It would be interesting to study the body distribution of B2 and determine whether this toxin-delivering PAA is able to escape liver capture.