Potent enzyme inhibitors derived from dromedary heavy-chain antibodies

Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.     

Polyanion inhibitors of HIV and other viruses. 7. Polyanionic compounds and polyzwitterionic compounds derived from cyclodextrins as inhibitors of HIV transmission

New polyanionic compounds were obtained from radical addition of thiomalic acid and mercaptopropionic acid onto perallylated cyclodextrins (CDs) under UV irradiation with a catalytic amount of alpha,alpha'-azobis(isobutyronitrile). All these polyanions, bearing 18-48 carboxylate groups, inhibited human immunodeficiency virus type 1 (HIV-1) strain IIIB replication in MT-4 cells at a 50% inhibitory concentration (IC50) of 0.1-2.9 microM, while not being toxic to the host cells at concentrations up to 62 microM. These compounds were also active against a clinical HIV-1 isolate (HE) at >/=4-fold higher concentrations. Only some compounds showed activity against the two HIV-2 strains (ROD and EHO) but at higher concentrations than those required to inhibit HIV-1 (IIIB and HE) replication. In addition, these compounds were not active against the M-tropic HIV-1 strain BaL but were active against simian immunodeficiency virus [SIV (MAC251)]. These compounds were also inhibitory to the replication of human cytomegalovirus at an IC50 of 1-10 microM, but not herpes simplex virus (type 1 and type 2) or other (picorna-, toga-, reo-, orthomyxo-, paramyxo-, bunya-, rhabdo-, and poxvirus) viruses. Radical addition on perallylated CDs of a protected cysteine gave polyzwitterionic compounds. None of these last compounds proved inhibitory to the replication of HIV-1, HIV-2, or any of the other viruses tested.     

Cyclic HIV-1 protease inhibitors derived from mannitol: synthesis, inhibitory potencies, and computational predictions of binding affinities

The molecular characteristics of midbrain dopamine (DA) neurons have been extensively studied in Parkinson's disease (PD). No such studies of the characteristics of midbrain DA neurons in Alzheimer's disease (AD) or Alzheimer's disease with parkinsonism (AD/Park) have been published. We examined the levels of tyrosine hydroxylase (TH) protein, and the expression of TH and dopamine transporter (DAT) mRNAs, in midbrain neurons of PD, AD, and AD/Park cases. In PD, the loss of TH protein in the ventral tier of the substantia nigra pars compacta (SNpc) of the PD group in accompanied by severe losses in the number of neurons that express TH mRNA and DAT mRNA (74% loss). Remaining neurons show a shift to higher concentrations of TH mRNA but a shift to lower concentrations of DAT mRNA per cell. Hence, there is evidence that compensation in the remaining neurons can elevate concentrations of TH mRNA and lower DAT mRNA. Alternatively, there may be a predilection for a loss of neurons with high levels of DAT mRNA and low TH mRNA levels within the SNpc of PD cases. There was no change in TH protein but an elevation of TH mRNA concentrations per neuron without any change in concentrations of DAT mRNA in the AD group. The AD/Park group did not exhibit changes in the level of TH protein, but showed a small loss (26%) of neurons in the SNpc and a greater loss in other regions of the midbrain (43-53%). Remaining DA neurons showed a marked shift to lower concentrations of DAT mRNA per neuron and a nonsignificant shift in cellular concentration of TH mRNA to higher levels. This is consistent with our previous work showing that with AD/Park there is a significant reduction in the number of DAT sites located on DA terminals in the striatum, but the midbrain neurons have not died. Our results indicate that the differential regulation of mRNAs encoding TH and DAT is similar in the parkinsonian disorders (PD and AD/Park) even though the degree of cell death is very different. This might suggest that compensatory events occur in these DA neurons in AD/Park that are similar to those in PD and that result in differential effects on mRNAs encoding TH and DAT proteins.     

Validation of protein models derived from experiment

The growing number of protein structures solved at atomic resolution holds the promise of further improvements in geometry-based validation parameters. Additionally, the estimated standard uncertainties of the atomic coordinates have been computed for a number of X-ray structures, providing a measure of the coordinate precision. In NMR spectroscopy, a measure analogous to the crystallographic R-factor has been developed.     

Development of hydroxyapatite derived from Indian coral

A simple method of converting the calcium carbonate skeleton of the corals available in the Indian coast into hydroxyapatite granules has been developed. By heating the coral to 900 degrees C, the organic materials were eliminated. Powder X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were employed to characterize the coral and to optimize the processing parameters as well as to confirm the hydroxyapatite formation. The coral used exhibits the presence of both aragonite and calcite phases (dimorphism). At a temperature of 900 degrees C the coral was found to decompose all the carbonate phases. The pre-heated coral is converted into hydroxyapatite by a chemical exchange reaction with di-ammonium phosphate under hydrothermal conditions. The hydroxyapatite obtained was in powder form and does not contain any impurities. The in vitro solubility test of the apatite granules performed in Gomoris, Michalelis, Sorensens, Ringer's and phosphate buffer of pH 7.2 and de-ionized water indicated the stability of the coralline hydroxyapatite.     

Substances derived from 4-de-N-methylfortimicin B

The preparation of 4-de-N-methylfortimicin A analogs as well as the preparation of 4-de-N-methyl-4-N-(beta-aminoethyl)-4-N-ethylfortimicin B is reported. It was shown that the 4-N-methyl group in fortimicin analogs is essential for antibacterial activity since neither the 4-de-N-methylfortimicin A nor the 4-de-N-methyl-4-N-(beta-aminoethyl)-4-N-ethylfortimicin B exhibited useful biological activity.     

Active metabolites derived from angiotensin II

Stasis of viscid secretions in cystic fibrosis (CF) leads to chronic infection, inflammation, and lung destruction. Chest physiotherapy (CPT) has been used for many years to assist in the removal of these secretions. However, the need for independently administered CPT exists, particularly for adolescents and the older CF patient. Two devices, the intrapulmonary percussive ventilator (IPV) and the Flutter device (Flutter) have been promoted for this purpose. This study compares these devices to standard, manual CPT. There was no difference in sputum quantity produced with any method studied. Transiently lower oxygen saturation was noted with standard CPT compared with the IPV and Flutter. Inconsistent but significant improvements in flow rates were noted with the two devices compared to standard CPT. Important trends to lower lung volumes, probably indicating decreased air trapping, were also noted with all three therapies at 1 and 4 hours after administration. There were no adverse effects with any treatment regimen. Larger and longer studies of these devices compared to standard CPT and with each other are warranted to assess their value for independent administration of CPT in CF patients and to determine long-term effects on maintenance of pulmonary function.     

A targeted library of small-molecule, tyrosine, and dual-specificity phosphatase inhibitors derived from a rational core design and random side chain variation

Tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosines while dual-specificity phosphatases (DSPases) dephosphorylate contiguous and semicontiguous phosphothreonine and phosphotyrosine on cyclin dependent kinases and mitogen-activated protein kinases. Consequently, PTPases and DSPases have a central role controlling signal transduction and cell cycle progression. Currently, there are few readily available potent inhibitors of PTPases or DSPases other than vanadate. Using a pharmacophore modeled on natural product inhibitors of phosphothreonine phosphatases, we generated a refined library of novel, phosphate-free, small-molecule compounds synthesized by a parallel, solid-phase combinatorial-based approach. Among the initial 18 members of this targeted diversity library, we identified several inhibitors of DSPases: Cdc25A, -B, and -C and the PTPase PTP1B. These compounds at 100 microM did not significantly inhibit the protein serine/threonine phosphatases PP1 and PP2A. Kinetic studies with two members of this library indicated competitive inhibition for Cdc25 DSPases and noncompetitive inhibition for PTP1B. Compound AC-alphaalpha69 had a Ki of approximately 10 microM for recombinant human Cdc25A, -B, and -C, and a Ki of 0.85 microM for the PTP1B. The marked differences in Cdc25 inhibition as compared to PTP1B inhibition seen with relatively modest chemical modifications in the modular side chains demonstrate the structurally demanding nature of the DSPase catalytic site distinct from the PTPase catalytic site. These results represent the first fundamental advance toward a readily modifiable pharmacophore for synthetic PTPase and DSPase inhibitors and illustrate the significant potential of a combinatorial-based strategy that supplements the rational design of a core structure by a randomized variation of peripheral substituents.     

Altered hemostasis in male rats following administration of the ACAT inhibitor SKF-99085

We have previously found that high-frequency stimulation of the medial amygdala (MeA) induces long-term potentiation (LTP) of the population spike in the perforant path-dentate granule cell synapses of anesthetized rats. In the present study, we investigated the influence of MeA stimulation on the relationship between the population excitatory postsynaptic potential (pEPSP) and population spike in the dentate gyrus. High-frequency stimulation of the MeA produced a leftward shift of the E-S curve, in which population spike amplitude was plotted against pEPSP slope at various stimulus intensities. MeA-induced population spike LTP was also observed under blockade of GABAergic inhibition with picrotoxin. These results suggests that MeA stimulation leads to a long-lasting change in the internal firing characteristics of the dentate granule cells.     

Ribonuclease inhibitors from mouse ascites cells

Ribonuclease inhibitors were found to be present attached to mouse ascites ribosomes and in the post-ribosomal supernatant. Both inhibitors inhibited pancreatic RNAses A and B and two endonucleases prepared from ascites cells but did not inhibit RNAses T1 or N1. Both inhibitors had the same sedimentation coefficient and this taken with the results above suggest that they are identical. The inhibitor was shown to interact directly with the RNAse itself.     

Tyrosinase inhibitors from Anacardium occidentale fruits

Anacardic acids, 2-methylcardols, and cardols isolated from various parts of the cashew [Anacardium occidentale] (Anacardiaceae) fruit have been found to exhibit tyrosinase inhibitory activity. Kinetic studies with the two principal active compounds, 6-[8(Z),11(Z),14-pentadecatrienyl]salicylic acid and 5-[8(Z),11(Z),14-pentadecatrienyl]resorcinol, have indicated that both of these phenolic compounds exhibit characteristic competitive inhibition of the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) by mushroom tyrosinase.     

Preparation and properties of mitochondria derived from synaptosomes

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

Effect of the ACAT inhibitor, HL-004, on cholesterol metabolism in macrophages

The effects of a novel acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor, HL-004, on cholesterol metabolism were examined in mice peritoneal macrophages. Cholesteryl ester-rich foam cells were induced by incubating macrophages with acetylated LDL. HL-004 prevented the accumulation of cholesteryl ester in the presence of the cholesterol acceptor, HDL. In the absence of HDL, HL-004 generated large amounts of free cholesterol in the cell. Moreover, HL-004 stimulated the efflux of cholesterol from preestablished foam cells in the presence of HDL. These results suggest that the inhibition of foam cell formation and the stimulation of foam cell regression by HL-004 are attributed to intracellular ACAT inhibition, and that HL-004 would be expected to exhibit an antiatherosclerotic effect through direct action on arterial wall.     

Binding pockets on the surface of human leukocyte elastase and human leukocyte cathepsin G. Implications to the design of inhibitors derived from human C-reactive protein

Analogs of the peptide Val-Thr-Val-Ala-Pro-Val-His-Ile, derived from the primary sequence of the acute phase reactant CRP, i.e. amino acid residues 89-96, were optimized to inhibit the enzymatic activities of human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG), which are associated with tissue damage occurring in the course of several chronic inflammatory conditions. hLE's major S1 pocket, lined mostly by hydrophobic amino acid residues, was shown by theoretical electrostatic potential calculations to possess some negative charge. This pocket was found to be extremely sensitive towards modifications in the P1 position of CRP derived inhibitors, with valine being the preferred amino acid. In contrast, the corresponding S1 pocket of hCG is large and accepts the positively charged 'aromatic' side chain of histidine, which increases most significantly the capability of CRP derived inhibitors. A prominent positive pocket was observed in the distant S7 region of hLE, which is generated by two exposed positive residues, Arg177 and Arg217, on the enzymes surface. This long range subsite was utilized to increase the hLE inhibitory activity of CRP derived peptide using the natural sequence of CRP, which contains a unique glutamic acid moiety in the P7 position. In contrast to the charged nature of hLE's S7 pocket, the corresponding pocket on the surface of hCG appears to be less prominent. Additional hydrophobic N-terminus modifications of CRP89-96 increased the inhibitory activity towards both enzymes, provided that residues P1 and p7, were designed according to the individual preferences of hLE and hCG. The unique interaction between the negative amino acid side chain of CRP with the positive S7 pocket of hLE as elucidated in this study, and additional subsite preferences may now be used in the design of novel therapeutic substances.     

Primary structure of trypsin inhibitors from Sicyos australis

Three trypsin inhibitors from Sicyos australis, have been isolated, purified and sequenced. Following protein extraction with ammonium sulphate, the mixture of inhibitors was separated from other proteins by trypsin-affinity chromatography. Subsequent purification of the individual inhibitors was accomplished by reversed-phase HPLC. The primary structures of each inhibitor were elucidated by a combination of protein sequencing and electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) on both the untreated and the reduced and S-carboxymethylated inhibitors. All three inhibitors show extensive sequence similarity with inhibitors from cultivated Cucurbitaceae species, although there are a number of novel residues present. One of the inhibitors has a blocked N-terminus (pyroglutamic acid) and the use of MS-MS was crucial to the elucidation of its primary structure. ESI-MS was further used to characterize the non-covalent complex between one of the inhibitors and trypsin.     

The hypolipidemic action of the ACAT inhibitor HL-004 in hamsters fed normal chow

1. A novel ACAT (acyl-CoA: cholesterol acyltransferase) inhibitor, HL-004, exhibited a strong inhibitory effect on the hepatic and intestinal ACAT, but was less effective on the adrenal ACAT in vitro. 2. HL-004 selectively decreased serum VLDL cholesterol, and inhibited hepatic ACAT activity in hamsters fed normal chow. 3. These results suggest that the cholesterol-lowering effect of HL-004 can be attributed to a decrease in hepatic VLDL secretion via inhibition of ACAT.     

Hydrolysis rates of pyrrolizidine alkaloids derived from Senecio jacobaea

BACKGROUND: Romano-Ward syndrome is a rare, autosomal-dominant cardiac conduction defect associated with syncope and life-threatening ventricular arrhythmia in children and adults. CASE: A fetus was noted to have moderate fetal bradycardia in the antenatal period. When studied postnatally, the child was found to have Romano-Ward syndrome. CONCLUSION: This is the first reported case of Romano-Ward syndrome diagnosed prenatally as fetal bradycardia. This report confirms that moderate fetal bradycardia does not indicate fetal distress, but it does indicate that fetuses should be studied for cardiac conduction defects in the neonatal period.