A comparative protease stability study of synthetic macrocyclic peptides that mimic two endocrine hormones

Peptide therapeutics have traditionally faced many challenges including low bioavailability, poor proteolytic stability and difficult cellular uptake. Conformationally constraining the backbone of a peptide into a macrocyclic ring often ameliorates these problems and allows for the development of a variety of new drugs. Such peptide-based pharmaceuticals can enhance the multi-faceted functionality of peptide side chains, permitting the peptides to bind cellular targets and receptors necessary to impart their role, while protecting them from degrading cellular influences. In the work described here, we developed three cyclic peptides, VP mimic1, VP mimic2 and OT mimic1, which mimic endocrine hormones vasopressin and oxytocin. Making notable changes to the overall structure and composition of the parent hormones, we synthesized the mimics and tested their durability against treatment with three proteases chosen for their specificity: pepsin, alpha-chymotrypsin, and pronase. Vasopressin and oxytocin contain a disulfide linkage leaving them particularly vulnerable to deactivation from the reducing environment inside the cell. Thus, we increased the complexity of our assays by adding reducing agent glutathione to each mixture. Subsequently, we discovered each of our mimics withstood protease treatment with less degradation and/or a slower rate of degradation as compared to both parent hormones and a linear control peptide.     

Insulin-like effects of vanadium: basic and clinical implications

Most mammalian cells contain vanadium at a concentration of about 20 nM, the bulk of which is probably in the reduced vanadyl (+4) form. Although this trace element is essential and should be present in the diet in minute quantities, no known physiological role for vanadium has been found thus far. In the late 1970s the vanadate ion was shown to act as an efficient inhibitor of Na+,K+-ATPase as well as of other related phosphohydrolases. In 1980 vanadium was reported to mimic the metabolic effects of insulin in rat adipocytes. During the last decade, vanadium has been found to act in an insulin-like manner in all three main target tissues of the hormone, namely skeletal muscles, adipose, and liver. Subsequent studies revealed that the action of vanadium salts is mediated through insulin-receptor independent alternative pathway(s). The investigation of the antidiabetic potency of vanadium soon ensued. Vanadium therapy was shown to normalize blood glucose levels in STZ-rats and to cure many hyperglycemia-related deficiencies. Therapeutic effects of vanadium were then demonstrated in type II diabetic rodents, which do not respond to exogenously administered insulin. Finally, clinical studies indicated encouraging beneficial effects. A major obstacle, however, is overcoming vanadium toxicity. Recently, several organically chelated vanadium compounds were found more potent and less toxic than vanadium salts in vivo. Such a newly discovered organic chelator of vanadium is described in this review.     

Potentiating vanadium-evoked glucose metabolism by novel hydroxamate derivatives

L-glutamic acid () monohydroxamate (L-Glu()HXM) enhances the insulinomimetic activity of vanadium ions both in vitro and in vivo. Based on this ligand as a lead compound, and in order to delineate molecular features relevant to its anti-diabetic potential, 14 related derivatives, including short peptides, were synthesized by solution as well as by solid phase methodologies. In addition, hydroxamate derivatives of (+) pantothenic acid and D-biotin were prepared. The vanadium binding capacity of the hydroxamates synthesized was apparent, yet each had a different ligand-ions stoichiometry. The in vitro lipogenic potency of several compounds toward rat adipocytes was demonstrated. Thus, vanadium complexes of L-Gln()HXM, L-Glu()HXM-Gly, L-Aad()HXM, di-Glu-,-HXM and of (+) pantothenic acid hydroxamate exhibited 82, 79, 76, 39 and 39% of maximal insulin activity, respectively. L-Aad()HXM, L-Glu()HXM-Gly and (+) pantothenic acid hydroxamate – by themselves – were found to possess 24, 14 and 10% of maximal insulin activity, respectively. In vivo potency, however, of L-Gln()HXM vanadium complex in streptozocin-treated rat diabetic model was less apparent.     

Potentiating vanadium-evoked glucose metabolism by novel hydroxamate derivatives

Summary L-glutamic acid (γ) monohydroxamate (L-Glu(γ)HXM) enhances the insulinomimetic activity of vanadium ions bothin vitro andin vivo. Based on this ligand as a lead compound, and in order to delineate molecular features relevant to its anti-diabetic potential, 14 related derivatives, including short peptides, were synthesized by solution as well as by solid phase methodologies. In addition, hydroxamate derivatives of (+) pantothenic acid and D-biotin were prepared. The vanadium binding, capacity of the hydroxamates synthesized was apparent, yet each had a different ligand-ions stoichiometry. Thein vitro lipogenic potency of several compounds toward rat adipocytes was demonstrated. Thus, vanadium complexes of L-Gln(α)HXM, L-Glu(γ)HXM-Gly, L-Aad(δ)HXM, di-Glu-γ,γ-HXM and of (+) pantothenic acid hydroxamate exhibited 82, 79, 76, 39 and 39% of maximal insulin activity, respectively. L-Aad (δ)HXM, L-Glu(γ)HXM-Gly and (+) pantothenic acid hydroxamate-by themselves —were found to possess 24, 14 and 10% of maximal insulin activity, respectively.In vivo potency, however, of L-Gln(α)HXM vanadium complex in streptozocin-treated rat diabetic model was less apparent.     

Vanadate restores glucose 6-phosphate in diabetic rats: a mechanism to enhance glucose metabolism

Vanadate mimics the metabolic actions of insulin. In diabetic rodents, vanadate also sensitizes peripheral tissues to insulin. We have analyzed whether this latter effect is brought about by a mechanism other than the known insulinomimetic actions of vanadium in vitro. We report that the levels of glucose 6-phosphate (G-6-P) in adipose, liver, and muscle of streptozotocin-treated (STZ)-hyperglycemic rats are 77, 50, and 58% of those in healthy control rats, respectively. Normoglycemia was induced by vanadium or insulin therapy or by phlorizin. Vanadate fully restored G-6-P in all three insulin-responsive peripheral tissues. Insulin did not restore G-6-P in muscle, and phlorizin was ineffective in adipose and muscle. Incubation of diabetic adipose explants with glucose and vanadate in vitro increased lipogenic capacity three- to fourfold (half-maximally effective dose = 11 +/- 1 microM vanadate). Lipogenic capacity was elevated when a threshold level of approximately 7.5 +/- 0.3 nmol G-6-P/g tissue was reached. In summary, 1) chronic hyperglycemia largely reduces intracellular G-6-P in all three insulin-responsive tissues; 2) vanadate therapy restores this deficiency, but insulin therapy does not restore G-6-P in muscle tissue; 3) induction of normoglycemia per se (i.e., by phlorizin) restores G-6-P in liver only; and 4) glucose and vanadate together elevate G-6-P in adipose explants in vitro and significantly restore lipogenic capacity above the threshold of G-6-P level. We propose that hyperglycemia-associated decrease in peripheral G-6-P is a major factor responsible for peripheral resistance to insulin. The mechanism by which vanadate increases peripheral tissue capacity to metabolize glucose and to respond to the hormone involves elevation of this hexose phosphate metabolite and the cellular consequences of this elevated level of G-6-P.