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We prepared a series of α-substituted malonester amides that were evaluated for their ability to inhibit acyl-CoA:cholesterol O-acyl transferase activity in vitro and to lower plasma total cholesterol levels in a variety of cholesterol-fed animal models. Compounds of this series were also useful in examining the relationship between adrenal toxicity and ACAT inhibition. One compound from this series, 9f, was a potent inhibitor of ACAT in both the microsomal and cellular assays. It was also bioavailable as determined by both a bioassay and a HPLC−UV assay. This compound was evaluated in both guinea pig and dog models of adrenal toxicity and compared to tetrazole amide 15. In the most sensitive species, the dog, both of these compounds achieved good plasma levels; however, compound 9f caused adrenal necrosis, whereas compound 15 had no effect on the adrenal gland. This adds to the growing body of evidence that the adrenal toxicity observed with ACAT inhibitors may not be mechanism related.

A series of diaryl-substituted heterocyclic ureas was prepared, and their ability to inhibit acyl-CoA:cholesterol O-acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in cholesterol-fed animal models in vivo was examined. N-(2,6-Diisopropylphenyl)-N‘-tetrazole- or isoxazole-substituted heterocyclic ureas proved optimal. A carbon chain of 11−14 carbons substituted 1,3 with respect to the amine provided the optimal side chain. Substitution of the alkyl chain generally lowered activity. Tetrazole urea 2i dosed at 3 mg/kg lowered plasma total cholesterol (TC) 67% in an acute, cholesterol-fed (C-fed) rat model of hypercholesterolemia and 47% in C-fed dogs. Tetrazole 2i, dosed at 10 mg/kg, also lowered TC 52% and raised HDL cholesterol 113% in rats with pre-established hypercholesterolemia.

A series of heterocyclic amides were synthesized and evaluated as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) in vitro and for cholesterol lowering in cholesterol-fed rats. Compounds were evaluated for cell-based macrophage ACAT inhibition, bioactivity, and adrenal toxicity. Candidates were selected for evaluation in cholesterol-fed dogs and, ultimately, the injured cholesterol-fed rabbit model of atherosclerosis. The heterocyclic amides potently inhibited rabbit liver ACAT (IC50's = 0.014−0.11 μM), and the majority of compounds significantly lowered plasma cholesterol (42−68%) in an acute cholesterol-fed rat model at 3 mg/kg. The most efficacious compounds in the rat were evaluated for bioactivity in vivo and arterial ACAT inhibition in a cell-based macrophage ACAT assay. Two highly bioactive analogs, (±)-2-(3-dodecylisoxazol-5-yl)-2-phenyl-N-(2,4,6-trimethoxyphenyl)acetamide (13a) and (±)-2-(5-dodecylisoxazol-3-yl)-2-phenyl-N-(2,4,6-trimethoxyphenyl)acetamide (16a), were selected for further study and were found to be nontoxic in a guinea pig model of adrenal toxicity. Compounds 13a and 16a lowered total cholesterol in the cholesterol-fed rat, rabbit, and dog models of pre-established hypercholesterolemia. Compound 13a in the injured cholesterol-fed rabbit model of atherosclerosis was effective in slowing the development of cholesteryl ester-rich thoracic aortic lesions, reducing lesion coverage by 53% at a dose of 1 mg/kg.

Background: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. Results: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II @b turn. Conclusions: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.

Several series of acyl-CoA:cholesterol O-acyltransferase inhibitors were prepared by the stepwise addition of nitrogen, oxygen, and sulfur nucleophiles to N-chlorosulfonyl isocyanate. The (aminosulfonyl)ureas 3−44 were the most potent inhibitors in vitro, with several compounds having IC50 values < 1 μM. Although the other series of compounds were not as potent in vitro, many compounds did display good in vivo activity in cholesterol-fed rats. Several of the oxysulfonyl carbamates (including CI-999, 115) showed excellent lipid-lowering activity in the chronic in vivo screen, demonstrating significant cholesterol lowering in a pre-established hypercholesterolemic state.

Tetrameric hemoglobin from the “fat innkeeper” worm Urechis caupo possesses a novel subunit arrangement having an “inside out” quaternary structure in that the G/H helices are located on the outer surface of the tetramer. A 5-A resolution crystal structure reveals that although the individual subunits are beta-like, having a distinct D helix and the general myoglobin fold, the subunit contacts are very different from those previously observed for hemoglobins. Furthermore, the hemoglobin from U. caupo is also quite different from the unusual hemoglobin tetramer from clam which also has its G/H helices on the outer surface but with the hemes in close proximity through E-F helical contacts (Royer, W. E., Jr., Love, W. E., and Fenderson, F. F. (1985) Nature 316, 277-280).

Crystals of the Fab' fragment from the monoclonal anti-peptide antibody B13I2 and of the Fab'-peptide antigen complex have been characterized. The monoclonal antibodies were raised against a synthetic homologue of the C-helix of myohemerythrin (residues 69–87 in myohemerythrin). The Fab'-peptide complex crystallizes in space group P6322 with unit cell dimensions a= b= 142.5 Å, c= 101.5 Å, α = β = 90°, γ = 120°, and Z= 1. The native Fab' crystallizes in space group P212121with unit cell dimensions a= 98.0 Å, b= 151.7 Å, c= 80.8 Å,α = β = γ = 90°, and Z= 2. Both crystal forms diffract to beyond 2.6 Å resolution. We also report the cDNA and predicted amino acid sequences for the variable regions of both the light and heavy chains of this anti-peptide antibody.