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The intense activity of phosphatidylinositol 3'-kinase (PtdIns 3'-kinase) results from the association of two phosphorylated tyrosines carrying YMXM sequences on insulin receptor substrate 1 with two carboxy SH2 domains of PtdIns 3'-kinase. Phosphotyrosine peptides carrying phosphorylated tyrosine YZPZ motifs in 3T3-L1 adipocytes exhibit enormous affinity for binding SH2 domains of PtdIns-3'kinase compared to those containing singular YZPZ sequence.

Tau pathology is associated with many neurodegenerative disorders, including Alzheimer's disease (AD), where the spatio-temporal pattern of tau neurofibrillary tangles strongly correlates with disease progression, which motivates therapeutics selective for misfolded tau. Here, we introduce a new avidity-enhanced, multi-epitope approach for protein-misfolding immunogen design, which is predicted to mimic the conformational state of an exposed epitope in toxic tau oligomers. A predicted oligomer-selective tau epitope 343 KLDFK 347 was scaffolded by designing a β-helix structure that incorporated multiple instances of the 16-residue tau fragment 339 VKSEKLDFKDRVQSKI 354 . Large-scale conformational ensemble analyses involving Jensen-Shannon Divergence and the embedding depth D showed that the multi-epitope scaffolding approach, employed in designing the β-helix scaffold, was predicted to better discriminate toxic tau oligomers than other "monovalent" strategies utilizing a single instance of an epitope for vaccine immunogen design. Using Rosetta, 10,000 sequences were designed and screened for the linker portions of the β-helix scaffold, along with a C-terminal stabilizing α-helix that interacts with the linkers, to optimize the folded structure and stability of the scaffold. Structures were ranked by energy, and the lowest 1% (82 unique sequences) were verified using AlphaFold. Several selection criteria involving AlphaFold are implemented to obtain a lead-designed sequence. The structure was further predicted to have free energetic stability by using Hamiltonian replica exchange molecular dynamics (MD) simulations. The synthesized β-helix scaffold showed direct binding in surface plasmon resonance (SPR) experiments to several antibodies that were raised to the structured epitope using a designed cyclic peptide. Moreover, the strength of binding of these antibodies to in vitro tau oligomers correlated with the strength of binding to the β-helix construct, suggesting that the construct presents an oligomer-like conformation and may thus constitute an effective oligomer-selective immunogen.

Oligomers of amyloid-β (AβO) are deemed key in synaptotoxicity and amyloid seeding of Alzheimer's disease (AD). However, the heterogeneous and dynamic nature of AβO and inadequate markers for AβO subtypes have stymied effective AβO identification and therapeutic targeting in vivo. We identified an AβO-subclass epitope defined by differential solvent orientation of the lysine 28 side chain in a constrained loop of serine-asparagine-lysine (cSNK), rarely displayed in molecular dynamics simulations of monomer and fibril ensembles. A mouse monoclonal antibody targeting AβO cSNK recognizes ∼50-60 kDa SDS-resistant soluble Aβ assemblages in AD brain and prolongs the lag phase of Aβ aggregation in vitro. Acute peripheral infusion of a murine IgG1 anti-AβO cSNK in two AD mouse models reduced soluble brain Aβ aggregates by 20-30%. Chronic cSNK peptide immunization of APP/PS1 mice engendered an anti-AβO cSNK IgG1 response without epitope spreading to Aβ monomers or fibrils and was accompanied by preservation of global PSD95 expression and improved cued fear memory. Our data indicate that the oligomer subtype AβO cSNK participates in synaptotoxicity and propagation of Aβ aggregation in vitro and in vivo.

Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final committed step in the biosynthesis of triglycerides. DGAT-1 knockout mice have been shown to be resistant to diet-induced obesity and have increased insulin sensitivity. Thus, inhibition of DGAT-1 may represent an attractive target for the treatment of obesity or type II diabetes. Herein, we report the discovery and characterization of a potent and selective DGAT-1 inhibitor PF-04620110 (3). Compound 3 inhibits DGAT-1 with an IC50 of 19 nM and shows high selectivity versus a broad panel of off-target pharmacologic end points. In vivo DGAT-1 inhibition has been demonstrated through reduction of plasma triglyceride levels in rodents at doses of ≥0.1 mg/kg following a lipid challenge. On the basis of this pharmacologic and pharmacokinetic profile, compound 3 has been advanced to human clinical studies.

A series of 2-phenoxy-3-phenylpropanoic acids has been prepared which contains many potent hypoglycemic agents as demonstrated by assessing glucose lowering in ob/ob mice. Some compounds (32, 33, 59) normalize plasma glucose in this diabetic model at doses of approximately 1 mg/kg. The mechanism of action of these drugs may involve enhanced glucose transport, especially in fat cells, but the compounds do not stimulate GLUT4 translocation and do not increase the levels of GLUT1 or GLUT4 in vivo. Thus, these compounds may enhance the intrinsic activity of the glucose transporter GLUT1 or GLUT4. Some compounds also modestly decrease hepatocyte gluconeogenesis in vitro, but this is not likely to be a major contributor to the hypoglycemic effect observed in vivo. Likewise, a modest decrease in food consumption observed with some of these compounds was shown by a pair-feeding experiment not to be the primary cause of the hypoglycemia observed.

The thiazolidinedione moiety of ciglitazone and its analogues can be replaced by an alpha-alkoxy or alpha-thioether carboxylic acid group. The influence of the nature of the R group, the length of the connector to the aromatic backbone of the molecule, and the stereochemistry have been studied. The most potent compounds have glucose-lowering activity at doses as low as 0.01 mg/kg.

A series of dihydrobenzofuran and dihydrobenzopyran thiazolidine-2,4-diones (compounds 3-26) was synthesized from the corresponding aryl aldehydes 1 in two steps. These compounds represent conformationally restricted analogues of the novel hypoglycemic ciglitazone. The series was evaluated by hypoglycemic effects in vitro by measuring stimulation of 2-deoxyglucose uptake in L6 myocytes and stimulation of expression of the glucose transporter protein in 3T3-L1 adipocytes. In vivo hypoglycemic effects were evaluated in the genetically obese ob/ob mouse, and structure-activity relationships are discussed. On the basis of this in vivo potency, we have selected the 2(R)-benzylbenzopyran derivative to be further studied in a clinical setting.

The reaction of hemin (Hm) with human hemopexin (Hx) has been studied in a mixed dimethyl sulfoxide (Me2SO)-water solvent system and in aqueous caffeine solutions. In both media, the kinetics could be described by a single, second-order process: (formula - see text) with k = 1.8 X 10(6) M-1 s-1 in 40% Me2SO-water [pH 7.4, mu = 0.2 M (NaCl)] and k = 3.9 X 10(7) M-1 s-1 in water [pH 7.4 mu = 0.2 M (NaCl), [caffeine] = 0.025 M]. The reaction shows an ionic strength dependence consistent with a residual 1+ to 2+ charge in the vicinity of the binding region of the protein. The kinetics of the transfer of hemin from albumin to hemopexin (formula - see text) were studied as a function of concentration, ionic strength, pH, and temperature. In experiments conducted at 3 less than or equal to [Alb]0/[Hx]0 less than or equal to 20 where the transfer kinetics are first order, k' = 5 X 10(-3) S-1 at mu = 0.3 M (NaCl), pH 7.1; the reaction is strongly dependent on ionic strength and choice of electrolyte. The addition of imidazole catalyzes this transfer process via a ligand-mediated pathway with k' = 5 X 10(-3) + 21[Im]T2. At [Alb]0/[Hx]0 = 92, the noncatalyzed transfer reaction is second order. From the kinetic analysis of the reaction under these conditions, an estimate is made of the distribution of hemin between the two proteins at concentration levels which are characteristic of serum. The association of hemin and hemopexin is approximately 30 times faster than that of hemin and albumin, a finding consistent with the recycling function of hemopexin during heme transport to the liver parenchymal cells.

The interactions of tetrakis(4-N-methylpyridyl)-porphine (H2TMpyP-4) and its copper(II), nickel(II), zinc(II), cobalt(III), iron(III), and manganese(III) derivatives with several nucleic acids have been investigated. Spectrophotometric titrations of H2TMpyP-4 and Cu(II)TMpyP-4 with the synthetic polymer poly(dG-dC) could be analyzed by a nearest-neighbor exclusion model leading to n approximately equal to two base pairs and equilibrium constants of 7.7 X 10(5) M-1 and 8.0 X 10(5) M-1, respectively. The other metal derivatives [except for the nickel(II) porphyrin] do not provide sufficiently large color changes with poly(dG-dC) to allow analysis. In contrast, all of these porphyrins interact with poly(dA-dT) and DNA. For those porphyrins investigated, the binding profiles are not adequately fit by a nearest-neighbor exclusion model but have profiles suggesting that cooperativity effects are important. Spectral and circular dichroic experiments both suggest base specificity. With calf thymus DNA, the copper(II) and nickel(II) derivatives show prominent negative circular dichroism (CD) features and large red shifts and hypochromicity of the Soret absorption band characteristic of GC specificity, as demonstrated with the synthetic polymer. The other metal derivatives show prominent positive induced visible CD features with small red shifts and hypochromicity of the absorption bands in the Soret region characteristic of AT specificity. Only the metal-free derivative has a conservative CD spectrum suggesting distribution among GC and AT sites.

Insulin stimulation of hexose transport in 3T3-L1 adipocytes was studied at 27 degrees C. At this temperature, the transport of 2-deoxyglucose was stimulated 8-fold, with a half-time of 9.5 min. Under the same conditions, the increase in cell surface glucose transporters, as measured by labeling in the intact cell with galactose oxidase and tritiated borohydride, was only 2.6-fold. Moreover, the half-times for the increase in cell surface glucose transporters and for the decrease in transporter number in the intracellular pool were both 4 min. Thus, these processes clearly precede the full stimulation of transport. These data are in agreement with immunolocalization studies of the glucose transporter in this cell line and further support the hypothesis that a second mechanism besides translocation is involved in the stimulation of hexose transport by insulin [Blok, J., Gibbs, E. M., Lienhard, G. E., Slot, J. W., & Gueze, H. J. (1988) J. Cell Biol. 106, 69-76]. The findings presented here indicate that neither the translocation of glucose transporters to, nor their subsequent insertion into, the plasma membrane is the rate-limiting step in the stimulation of hexose transport by insulin. Rather, there is a second mechanism of activation, which is rate limiting and occurs after the transporter is in the plasma membrane.

The transfer of hemin from one protein to another is an event biologically important for the conservation of heme iron. Hemin entering the circulation (or added to serum) is mainly bound by albumin and then transferred to hemopexin [Morgan, W.T., Liem, H.H., Sutor, R.P., & Muller-Eberhard, U. (1976) Biochim. Biophys. Acta 444, 435-445], and we are now investigating which mechanisms may be operative in enhancing this process. The presence of imidazole has been demonstrated to accelerate hemin transfer from albumin to hemopexin [Pasternack, R.F., Gibbs, E.J., Hoeflin, E., Kosar, W.P., Kubera, G., Skowronek, C. A., Wong, N.M., & Muller-Eberhard, U. (1983) Biochemistry 22, 1753-1758]. The present work is an examination of the effect of the reduction of albumin-bound hemin on the rate of its transfer to hemopexin. Hemin (HmIII., ferriprotoporphyrin IX) was reduced to HmII (ferroprotoporphyrin IX) by the addition of sodium dithionite under argon. The reduction kinetics of HmIII to HmII were studied separately in the two complexes: with human serum albumin (HSA), which binds up to 20 mol of heme/mol (the first mole with K congruent to 10(8)), and with hemopexin (HHx), which binds heme equimolarly (K congruent to 10(13)). The rate of reduction of HmIII to HmII on HSA was first order over several half-lives and linearly dependent on [S2O4(2-)]1/2. At [HSA]0/[HmIII] = 3, the kobsd was (5 X 10(-3) + 0.75[S2O4(2-)]1/2, and with [HSA]/[HmIII] approximately 25, the kobsd was (2 X 10(-3)) + 0.25[S2O4(2-)]1/2. The reduction of HmIII to HmII on human hemopexin (HHx) is much more rapid with kobsd = (2.5 X 10(3))[S2O4(2-)]1/2.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetrakis(4-7V-methylpyridyl)porphine (H2TMpyP) and a number of its metal derivatives interact extensively with mononucleotides and mononucleosides in aqueous solution. The complexes formed are of a stacking-type involving extensive overlap of the -systems of the porphyrin and purine or pyrimidine bases. Coulombic attractions help stabilize the complexes but there is no evidence for ligation of the bases to axial sites of the metalloporphyrins. Stability constants determined via NMR and spectrophotometric titrations are larger for purine bases than pyrimidines with a given porphyrin derivative. More dramatic influences on stability result from changing porphyrins. Porphyrins having no axial ligands (e.g., metal-free copper(II), palladium(II), and nickel(II) derivatives) or one axial ligand (Zn(II)) produce much larger interactions with a given nucleotide or nucleoside than do metalloporphyrins having two axial ligands (e.g., Mn(III), Fe(III), or Co(III)). The kinetics of the interaction of H2TMpyP with 2'-deoxyadenosine S'-monophosphate (dAMP) were studied via the laser raman temperature-jump method. The measured rate constants are consistent with a simple stacking model for the interaction.