14 research outputs found
High-Yield Synthesis of Human Insulin-Like Peptide 5 Employing a Nonconventional Strategy
A simplified
route to synthesis of INSL5 is reported, where the
elimination of intermediate purification steps and nonconventional
disulfide pairing results in final yields that are an order of magnitude
higher than in previously reported stepwise syntheses. The intramolecular
disulfide of A-chain was produced by a thiol displacement of StBu-protected
cysteine, and was followed by an A-B chain disulfide formation in
dimethylsulfoxide (DMSO). The final disulfide was formed by deprotection
of StBu-cysteines in hydrofluoric acid (HF) at room temperature, which
is a historical approach infrequently employed today, followed by
oxidation using 2,2-dithiobisÂ(5-nitropyridine) (DTNP) in acidic aqueous
buffer. Throughout the synthesis, an isoacyl surrogate to a midsequence
native amide bond was utilized to enhance solubility of the intermediate
compounds
Chemical Synthesis of Insulin Analogs through a Novel Precursor
Insulin remains a challenging synthetic
target due in large part
to its two-chain, disulfide-constrained structure. Biomimetic single
chain precursors inspired by proinsulin that utilize short peptides
to join the A and B chains can dramatically enhance folding efficiency.
Systematic chemical analysis of insulin precursors using an optimized
synthetic protocol identified a 49 amino acid peptide named DesDi,
which folds with high efficiency by virtue of an optimized structure
and could be proteolytically converted to bioactive two-chain insulin.
In subsequent applications, we observed that the folding of the DesDi
precursor was highly tolerant to amino acid substitution at various
insulin residues. The versatility of DesDi as a synthetic insulin
precursor was demonstrated through the preparation of several alanine
mutants (A10, A16, A18, B12, B15), as well as ValA16, an analog that
was unattainable in prior reports. <i>In vitro</i> bioanalysis
highlighted the importance of the native, hydrophobic residues at
A16 and B15 as part of the core structure of the hormone and revealed
the significance of the A18 residue to receptor selectivity. We propose
that the DesDi precursor is a versatile synthetic intermediate for
the preparation of diverse insulin analogs. It should enable a more
comprehensive analysis of function to insulin structure than might
not be otherwise possible through conventional approaches
Synthesis of Four-Disulfide Insulin Analogs via Sequential Disulfide Bond Formation
Naturally
occurring, multiple cysteine-containing peptides are
a structurally unique class of compounds with a wide range of therapeutic
and diagnostic applications. The development of reliable, precise
chemical methods for their preparation is of paramount importance
to facilitate exploration of their utility. We report here a straightforward
and effective approach based on stepwise, sequentially directed disulfide
bond formation, exemplified by the synthesis of four-disulfide bond-containing
insulin analogs. Cysteine protection consisted of <i>tert</i>-butylthiol (S<i>t</i>Bu), thiol-trimethoxyphenyl (STmp),
trityl (Trt), 4-methoxytrityl (Mmt), S-acetamidomethyl (Acm), and <i>tert</i>-butyl (<i>t</i>Bu). This report describes
chemistry that is broadly applicable to cysteine-rich peptides and
the influence of a fourth disulfide bond on insulin bioactivity
Discovery of High Potency, Single-Chain Insulin Analogs with a Shortened B‑Chain and Nonpeptide Linker
A series of novel, single chain insulin
analogs containing polyethylene
glycol based connecting segments were synthesized by native chemical
ligation and tested for biological activity. While the full length
single chain insulin analogs exhibited low potency, deletion of amino
acids B26–B30 unexpectedly generated markedly higher activity.
This observation is unprecedented in all previous studies of single
chain insulin analogs and is consistent with the presumption that
in the native hormone this sequence must translocate to achieve high
potency insulin receptor interaction. Optimization of the sequence
yielded an insulin analog with potency and selectivity comparable
to that of native insulin. These results establish a basis for discovery
of novel higher potency, single chain insulin analogs of shortened
length
Discovery of a potent GIPR peptide antagonist that is effective in rodent and human systems
Objective: Glucose-dependent insulinotropic polypeptide (GIP) is one of the two major incretin factors that regulate metabolic homeostasis. Genetic ablation of its receptor (GIPR) in mice confers protection against diet-induced obesity (DIO), while GIPR neutralizing antibodies produce additive weight reduction when combined with GLP-1R agonists in preclinical models and clinical trials. Conversely, GIPR agonists have been shown to promote weight loss in rodents, while dual GLP-1R/GIPR agonists have proven superior to GLP-1R monoagonists for weight reduction in clinical trials. We sought to develop a long-acting, specific GIPR peptide antagonist as a tool compound suitable for investigating GIPR pharmacology in both rodent and human systems. Methods: We report a structure–activity relationship of GIPR peptide antagonists based on the human and mouse GIP sequences with fatty acid-based protraction. We assessed these compounds in vitro, in vivo in DIO mice, and ex vivo in islets from human donors. Results: We report the discovery of a GIP(5-31) palmitoylated analogue, [Nα-Ac, L14, R18, E21] hGIP(5-31)-K11 (γE-C16), which potently inhibits in vitro GIP-mediated cAMP generation at both the hGIPR and mGIPR. In vivo, this peptide effectively blocks GIP-mediated reductions in glycemia in response to exogenous and endogenous GIP and displays a circulating pharmacokinetic profile amenable for once-daily dosing in rodents. Co-administration with the GLP-1R agonist semaglutide and this GIPR peptide antagonist potentiates weight loss compared to semaglutide alone. Finally, this antagonist inhibits GIP- but not GLP-1-stimulated insulin secretion in intact human islets. Conclusions: Our work demonstrates the discovery of a potent, specific, and long-acting GIPR peptide antagonist that effectively blocks GIP action in vitro, ex vivo in human islets, and in vivo in mice while producing additive weight-loss when combined with a GLP-1R agonist in DIO mice
The islet-expressed Lhx1 transcription factor interacts with Islet-1 and contributes to glucose homeostasis
Modulation of cytokine responses of murine CD8+ αβ intestinal intraepithelial lymphocytes by IL-4 and IL-12
Chemical hybridization of glucagon and thyroid hormone optimizes therapeutic impact for metabolic disease
Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease