Bohumír Grüner

From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3′ subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.

Interaction of fluorescently substituted metallacarboranes with cyclodextrins and phospholipid bilayers: fluorescence and light scattering study.

We prepared two fluorescein-[3-cobalt(III) bis(1,2-dicarbollide)](-) conjugates. They are sparingly soluble in water and form large aggregates in aqueous solutions. An extensive study on their spectral and aggregation behavior was carried out. To prepare their well-defined dispersion in aqueous systems, we studied the interaction of both probes with two biocompatible amphiphilic systems, cyclodextrins, which are frequently used in drug-delivery systems, and phospholipid membranes, which are the major constituents of cell barriers in living organisms. The presence of fluorescein in both conjugates allows us to study their behavior in detail by steady-state and time-resolved fluorometry, fluorescence correlation spectroscopy, and fluorescence lifetime imaging. The self-assembly of these metallacarboranes in aqueous solutions was studied by dynamic light scattering. The study shows that the compounds interact with cyclodextrins that increases their solubility in water, and they solubilize easily in phospholipid bilayers.

Liquid chromatographic resolution of enantiomers of deltahedral carborane and metallaborane derivatives

Abstract A successful liquid chromatographic separation of twelve enantiomeric pairs of zwitterionic eleven-vertex nido -carboranes of the type L-R-7,8-C 2 B 9 H 10 (L = Me 2 S, Me 2 SCH 2 , C 6 H 5 N, C 6 H 5 NCH 2 ; R = H, CH 3 , C 6 H 5 , Me = methyl) and of the 4-MeS-3-C 5 H 5 -1,2,3-C 2 CoB 9 H 10 mixed sandwich complex on β-cyclodextrin (CD) chiral stationary phases (CSP) in aqueous-organic mobile phases is described. A comparison of two β-CD CSP columns. Tessek β-CD and Astec Cyclobond I, differing in enantioselectivity and resolution of individual compounds (to some extent), is presented together with a study of the factors controlling retention and enantiomeric resolution.

Micelle-like nanoparticles of block copolymer poly(ethylene oxide)-block-poly(methacrylic acid) incorporating fluorescently substituted metallacarboranes designed as HIV protease inhibitor interaction probes.

We prepared nanoparticles differing in morphology from double-hydrophilic block copolymer poly(ethylene oxide)-block-poly(methacrylic acid), PEO-PMA, and two types of fluorescein-[3-cobalt(III) bis(1,2-dicarbollide)] conjugates, GB176 and GB179, in alkaline buffer. GB176 molecule consists of fluorescein attached to the metallacarborane anion. In GB179 molecule, the fluorescein moiety connects two metallacarborane anions. The self-assembly is based on the unusual interaction of metallacarborane clusters with PEO blocks which form insoluble micellar cores. The GB176 containing nanoparticles are loose and irregular, while the GB179 ones are rigid and spherical. The structure of nanoparticles depends to some extent on a procedure of preparation. The micelles were studied by static and dynamic light scattering, fluorometry and atomic force microscopy. Since the metallacarborane conjugates act as potent inhibitors of HIV protease, the presented system is important from the point of view of drug delivery.

Exploration of the electrophoretic behaviour of borane cluster anions and of the capability of capillary electrophoresis to separate them chirally

Mobilities of investigated boron cluster compounds in 3-(N-morpholino)propanesulfonic and phosphate buffers adjusted to pH 7 either with sodium hydroxide or with tris(hydroxymethyl)aminomethane depend on both buffer ions. The zone width and zone asymmetry, which are usually markedly higher than those of organic or common inorganic ions of comparable size, depend on the type of the borane cluster anion. Unusual shapes of zones of two investigated compounds have been found in tris phosphate buffer. Acetonitrile was superior to methanol as an organic additive to separation systems from the viewpoint of the zone symmetry and separation speed. Narrow trigonal zones, typical of organic ions non-interacting with the capillary wall, have been observed for some bridged sandwich cobalt complexes in run buffers with the addition of acetonitrile. The interaction of borane cluster anions with beta-cyclodextrin cavity is excessively strong in purely aqueous solutions. Methanol and acetonitrile, which generally weaken the interaction, sometimes affect the separation enantioselectivity of various compounds in different ways in addition to the weakening effect. Chiral discrimination was reached for all ten investigated anions, which belong to four different structural types of cluster boranes. Stability constants estimated for some analyte-beta-cyclodextrin complexes range between 100 and 1800 l/mol in acceptable separations. The relative difference of the constants was from 3 to 20%.

Emerging subject for chiral separation science: cluster boron compounds.

The structural chirality is an inherent feature of fully synthetic boron cluster compounds that sometimes exhibit unique biochemical effects. HPLC studies with zwitter-ionic cluster boron compounds and electrophoretic studies with boron cluster anions reveal that the chiral separability of these species is remarkably dissimilar to that of organic species, if uncharged cyclodextrins are used as chiral selectors. Furthermore, marked differences were found between the analytical characteristics of the chiral separations of the boron cluster species and those of the organic species with uncharged cyclodextrins. The present-day experimental database indicates that the rules valid for the chiral separations of the organic species cannot be applied to the chiral separations of the boron cluster species without experimental verification. The current extent of research work devoted to the investigation of chirality and chiral separations of boron cluster species is negligibly small in comparison with that devoted to the investigation of chirality and chiral separations of organic species. This makes difficult a reliable explanation of both the particularities observed in chiral separations of boron cluster species with cyclodextrins as chiral selectors and the strange effects related to these separations at the moment.

Nitric oxide synthases activation and inhibition by metallacarborane- cluster-based isoform-specific affectors

A small library of boron-cluster- and metallacarborane-cluster-based ligands was designed, prepared, and tested for isoform-selective activation or inhibition of the three nitric oxide synthase isoforms. On the basis of the concept of creating a hydrophobic analogue of a natural substrate, a stable and nontoxic basic boron cluster system, previously used for boron neutron capture therapy, was modified by the addition of positively charged moieties to its periphery, providing hydrophobic and nonclassical hydrogen bonding interactions with the protein. Several of these compounds show efficacy for inhibition of NO synthesis with differential effects on the various nitric oxide synthase isoforms.

Chiral resolution of enantiomers of asymmetric cobaltacarboranes with a monoatomic bridge between ligands by liquid chromatography on a β-cyclodextrin column

Abstract The HPLC resolution on a β-cyclodextrin column is reported of thirteen enantiomeric pairs of racemates of the type 6, 6′-μ-R-E(1,7-C2B9H10)2-2-Co with oxygen, sulphur and nitrogen bridges (E) and with a variety of R substituents differing in size and electronic properties along with structural factors influencing the capacity factors, selectivity and resolution of individual compounds. A semi-preparative method for chiral separations of these racemates is described and circular dichroism (CD) spectra of several pure enantiomers are presented. The CD spectra indicate the same absolute configurations of all enantiomers eluted first or last independent of the nature of the bridging atom and the size of the bridge substituting group R.

Chromatographic behaviour of the closo-[B12H12]2- derivatives on hydroxyethylmethacrylate gels

Abstract Halo derivatives (Cl, Br, I) of the closo -dodecahydro-dodecaborate anion, [B 12 H 12 ] 2− , and their positional isomers were separated on Separon HEMA hydroxyethylmetacrylate sorbents. Different effects on the separation process, including cations and anions in the mobile phase, the electrolyte concentration in the eluent, pH, addition of organic modifier and separation temperature, were studied. Optimum conditions for the separation were accomplished with Separon HEMA BIO 300 and 1000 sorbents using 0.1–0.5 M sodium perchlorate solutions in 0.01 M phosphate buffer (pH 8.5) as the mobile phase. The separated compounds were detected using direct UV detection in the range 200–210 nm.

Radiolysis of C5-BTBP in cyclohexanone irradiated in the absence and presence of an aqueous phase

Abstract Spent nuclear fuel contains many highly radioactive species; hence solvents used in reprocessing will be subjected to radiolysis. In this study, solvents containing one of the BTBP molecules intended for the separation of trivalent actinides and lanthanides, the so called C5-BTBP, have been subjected to radiolysis and hydrolysis. We present here that this compound shows a dramatic decrease in both distribution ratios and separation factor when irradiated with higher doses up to 50 kGy; particularly in the presence of an aqueous phase. Furthermore, fast hydrolytic degradation is observed, which significantly contributes to the overall degree of decomposition. This is supported by speciation studies performed by HPLC and LC-MS methods. Proposed structures of the highest-yield degradation products are presented and they seem to confirm previously drawn structures for these products. From these studies it can be concluded that the presence of nitric acid or nitrate during irradiation leads to higher content of species containing keto groups.