Exploratory studies on soluble small molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics, which inhibit the interaction of gp120 with CD4, have been developed. Original CD4 mimics such as NBD-556, which has an aromatic ring, an oxalamide linker and a piperidine moiety, possess significant anti-HIV activity but with their hydrophobic aromatic ring-containing structures are poorly soluble in water. We have developed derivatives with a halopyridinyl group in place of the phenyl group, such as KKN-134, and found them to have excellent aqueous solubility. Other leads that were examined are YIR-821, a compound with a cyclohexane group in a spiro attachment to a piperidine ring and a guanidino group on the piperidine nitrogen atom, and its PEGylated derivative, TKB-002. YIR-821 and TKB-002 retain potent anti-HIV activity. Here, new CD4 mimics, in which the phenyl group was replaced by a halopyridinyl group with the halogen atoms in different positions, their derivatives without a cyclohexane group on the piperidine ring and their hybrid molecules with PEG units were designed and synthesized. Some of these compounds show significantly higher aqueous solubility with maintenance of certain levels of anti-HIV activity. The present data should be useful in the future design of CD4 mimic molecules.

Soluble-type small-molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics have been reported previously as HIV-1 entry inhibitors, which block the interaction between the Phe43 cavity of HIV-1 gp120 and the host CD4. Known CD4 mimics such as NBD-556 possess significant anti-HIV activity but are less soluble in water, perhaps due to their hydrophobic aromatic ring-containing structures. Compounds with a pyridinyl group in place of the phenyl group in these molecules have been designed and synthesized in an attempt to increase the hydrophilicity. Some of these new CD4 mimics, containing a tetramethylpiperidine ring show significantly higher water solubility than NBD-556 and have high anti-HIV activity and synergistic anti-HIV activity with a neutralizing antibody. The CD4 mimic that has a cyclohexylpiperidine ring and a 6-fluoropyridin-3-yl ring has high anti-HIV activity and no significant cytotoxicity. The present results will be useful in the future design and development of novel soluble-type molecule CD4 mimics.

Metabolic engineering of mevalonate-producing Escherichia coli strains based on thermodynamic analysis.

Thermodynamic states of the central metabolism in a metabolically engineered Escherichia coli strain producing mevalonate (MVA) were studied to identify metabolic reactions with regulatory function for improvement of the specific rate of MVA production. Intracellular concentrations of metabolites were determined for E. coli strains expressing Enterococcus faecalis genes mvaE and mvaS (strain MV) by gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS). Mixtures of 13C-labeled metabolites served as internal standards were prepared from E. coli cultured in a completely 13C-labeled medium. Based on the concentration data, the change in Gibbs energy (ΔG) and substrate saturation ([S]/KM) were calculated for each metabolic reaction and then compared between the control and MVA-producing strains. The thermodynamic and kinetic analyses showed that further activation of thermodynamically feasible reactions in the upper part of glycolysis and the pentose phosphate pathway seems difficult and that metabolic bypassing to the Entner-Doudoroff pathway was a promising strategy to improve the acetyl coenzyme A (AcCoA) and NADPH supply required for MVA biosynthesis. Strain MV-ΔGndΔGntR was constructed by deletion of the gnd and gntR genes, which respectively encode 6-phosphogluconate dehydrogenase and a negative regulator of the expression of two enzyme genes responsible for the Entner-Doudoroff pathway. Cultivation in the nongrowth phase revealed that the yield and specific production rate of MVA increased to 0.49 ±â€¯0.01 Cmol (Cmol glucose)-1 and 2.61 ±â€¯0.10 mmol (g dry cell weight)-1 h-1, which were 113% and 158% that of the MV strain, respectively.

Hybrids of Small-Molecule CD4 Mimics with Polyethylene Glycol Units as HIV Entry Inhibitors.

CD4 mimics are small molecules that inhibit the interaction of gp120 with CD4. We have developed several CD4 mimics. Herein, hybrid molecules consisting of CD4 mimics with a long alkyl chain or a PEG unit attached through a self-cleavable linker were synthesized. In anti-HIV activity, modification with a PEG unit appeared to be more suitable than modification with a long alkyl chain. Thus, hybrid molecules of CD4 mimics, with PEG units attached through an uncleavable linker, were developed and showed high anti-HIV activity and low cytotoxicity. In investigation of pharmacokinetics in a rhesus macaque, a hybrid compound had a more effective PK profile than that of the parent compound, and intramuscular injection was a more useful administration route to maintain the high blood concentration of the CD4 mimic than intravenous injection. The presented hybrid molecules of CD4 mimics with a PEG unit would be practically useful when combined with a neutralizing antibody.

Metabolic impact of nutrient starvation in mevalonate-producing Escherichia coli.

The aim of this work was to enhance mevalonate yield from glucose in Escherichia coli by essential nutrient starvations and to reveal these effects on the central carbon metabolism. Stationary phase culture without essential nutrients such as nitrogen, sulfur, and magnesium was evaluated using an engineered E. coli introducing mvaE and mvaS genes from Enterococcus faecalis. Sulfur starvation resulted in the highest mevalonate yield of 0.61C-molC-mol-1 from glucose. The metabolic impacts of nutrient starvation were investigated by 13C-metabolic flux analysis. Under nitrogen starvation, the flux of the TCA cycle was large, causing high CO2 production. This was caused by degradation of mevalonate synthesis pathway enzymes. Under magnesium starvation, NADPH production was decreased, which limited mevalonate synthesis and promoted an overflow of acetate. Sulfur starvation not only suppressed the TCA cycle flux, but also supplied NADPH for mevalonate synthesis.

Fluoropolymer Nanosheet as a Wrapping Mount for High-Quality Tissue Imaging.

In the field of biological microscopy technology, it is still a practical challenge to obtain high-quality tissue images, due to the tissue desiccation that occurs during observations without an effective sample mounting. Inspired by the use of plastic food wrap, this study proposes the use of polymer thin films (also known as nanosheets) to fix the tissue samples. Water-repellent nanosheets composed of the amorphous fluoropolymer CYTOP are prepared with adjustable thicknesses and their hydrophobicity, transparency, and adhesion strength are evaluated. They show excellent water-retention effect and work well for sample fixation. By wrapping cleared mouse brain slices with a 133 nm thick CYTOP nanosheet, this study achieves high spatial resolution neuron images while scanning over a large area for a long period of time. No visible artifacts arising from sample shrinkage can be detected. This study also expects that nanosheet wrapping could be effective over a longer time span by combination with conventional agarose embedding.