Fa Liu

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.

Chemical synthesis of peptides within the insulin superfamily.

The synthesis of insulin has inspired fundamental advances in the art of peptide science while simultaneously revealing the structure–function relationship of this centrally important metabolic hormone. This review highlights milestones in the chemical synthesis of insulin that can be divided into two separate approaches: (i) disulfide bond formation driven by protein folding and (ii) chemical reactivity‐directed sequential disulfide bond formation. Common to the two approaches are the persistent challenges presented by the hydrophobic nature of the individual A‐chain and B‐chain and the need for selective disulfide formation under mildly oxidative conditions. The extension and elaboration of these synthetic approaches have been ongoing within the broader insulin superfamily. These structurally similar peptides include the insulin‐like growth factors and also the related peptides such as relaxin that signal through G‐protein‐coupled receptors. After a half‐century of advances in insulin chemistry, we have reached a point where synthesis is no longer limiting structural and biological investigation within this family of peptide hormones. The future will increasingly focus on the refinement of structure to meet medicinal purposes that have long been pursued, such as the development of a glucose‐sensitive insulin. Copyright

Concise Synthetic Routes to Human Insulin

We report a set of concise and efficient routes for the chemical synthesis of human insulin using a two- or three-step combination procedure that employs Trt, Acm, and t-Bu cysteine protection schemes. Starting with resin-bound assembled A and B chains, human insulin can be obtained within the span of a single work day in 5.4% overall yield based on the crude A or B chain.

Irreversible Sortase A-Mediated Ligation Driven by Diketopiperazine Formation

Sortase A (SrtA)-mediated ligation has emerged as an attractive tool in bioorganic chemistry attributing to the remarkable specificity of the ligation reaction and the physiological reaction conditions. However, the reversible nature of this reaction limits the efficiency of the ligation reaction and has become a significant constraint to its more widespread use. We report herein a novel set of SrtA substrates (LPETGG-isoacyl-Ser and LPETGG-isoacyl-Hse) that can be irreversibly ligated to N-terminal Gly-containing moieties via the deactivation of the SrtA-excised peptide fragment through diketopiperazine (DKP) formation. The convenience of the synthetic procedure and the stability of the substrates in the ligation buffer suggest that both LPETGG-isoacyl-Ser and LPETGG-isoacyl-Hse are valuable alternatives to existing irreversible SrtA substrate sequences.

An Iodine-Free and Directed-Disulfide-Bond-Forming Route to Insulin Analogues

An iodine-free synthetic route to insulin analogues has been established via a directed disulfide bond formation strategy. This method is completely compatible with oxidation-sensitive residues. The key step is constructing the third disulfide bond via a novel procedure involving phenylacetylaminomethyl group (Phacm), immobilized Penicillin G Acylase, and Ellmans reagent. We expect that this method could be broadly utilized for synthesizing insulin-like and other cysteine-rich peptides, in particular, where oxidation-sensitive residues are present in the sequence.

An Fmoc compatible, O to S shift-mediated procedure for the preparation of C-terminal thioester peptides.

We report a practical 2-hydroxy-3-mercapto-propionic acid (Hmp)/2-methylpiperidine (2-MP) based Fmoc chemistry procedure to prepare the C-terminal Hmp peptides, which serve as the precursors of C-terminal thioester peptides. The subsequent O to S acyl shift and thiol-exchange mediated thioester conversion of the crude precursor peptides can be accomplished smoothly under mild conditions to provide the desired thioester peptides with good yield and high quality. This is a highly adaptable approach, and we envision its broad application in the preparation of C-terminal thioester peptides.

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 tert-butylthiol (StBu), thiol-trimethoxyphenyl (STmp), trityl (Trt), 4-methoxytrityl (Mmt), S-acetamidomethyl (Acm), and tert-butyl (tBu). This report describes chemistry that is broadly applicable to cysteine-rich peptides and the influence of a fourth disulfide bond on insulin bioactivity.

Synthetic Advances in Insulin-like Peptides Enable Novel Bioactivity

Insulin is a miraculous hormone that has served a seminal role in the treatment of insulin-dependent diabetes for nearly a century. Insulin resides within in a superfamily of structurally related peptides that are distinguished by three invariant disulfide bonds that anchor the three-dimensional conformation of the hormone. The additional family members include the insulin-like growth factors (IGF) and the relaxin-related set of peptides that includes the so-called insulin-like peptides. Advances in peptide chemistry and rDNA-based synthesis have enabled the preparation of multiple insulin analogues. The translation of these methods from insulin to related peptides has presented unique challenges that pertain to differing biophysical properties and unique amino acid compositions. This Account presents a historical context for the advances in the chemical synthesis of insulin and the related peptides, with division into two general categories where disulfide bond formation is facilitated by native conformational folding or alternatively orthogonal chemical reactivity. The inherent differences in biophysical properties of insulin-like peptides, and in particular within synthetic intermediates, have constituted a central limitation to achieving high yield synthesis of properly folded peptides. Various synthetic approaches have been advanced in the past decade to successfully address this challenge. The use of chemical ligation and metastable amide bond surrogates are two of the more important synthetic advances in the preparation of high quality synthetic precursors to high potency peptides. The discovery and application of biomimetic connecting peptides simplifies proper disulfide formation and the subsequent traceless removal by chemical methods dramatically simplifies the total synthesis of virtually any two-chain insulin-like peptide. We report the application of these higher synthetic yield methodologies to the preparation of insulin-like peptides in support of exploratory in vivo studies requiring a large quantity of peptide. Tangentially, we demonstrate the use of these methods to study the relative importance of the IGF-1 connecting peptide to its biological activity. We report the translation of these finding in search of an insulin analog that might be comparably enhanced by a suitable connecting peptide for interaction with the insulin receptor, as occurs with IGF-1 and its receptor. The results identify a unique receptor site in the IGF-1 receptor from which this enhancement derives. The selective substitution of this specific IGF-1 receptor sequence into the homologous site in the insulin receptor generated a chimeric receptor that was equally capable of signaling with insulin or IGF-1. This novel receptor proved to enhance the potency of lower affinity insulin ligands when they were supplemented with the IGF-1 connecting peptide that similarly enhanced IGF-1 activity at its receptor. The chimeric insulin receptor demonstrated no further enhancement of potency for native insulin when it was similarly prepared as a single-chain analogue with a native IGF-1 connecting peptide. These results suggest a more highly evolved insulin receptor structure where the requirement for an additional structural element to achieve high potency interaction as demonstrated for IGF-1 is no longer required.

Site-specific fluorescein labeling of human insulin.

Three fluorescein derivatives of human insulin (HI, 1) labeled at positions NαA1, NαB1 and NεB29 respectively, were synthesized using an N‐trifluoroacetyl‐based protecting group scheme. The Tfa protecting group introduced by reaction with ethyl trifluoroacetate was found to be stable in aqueous and organic media and efficiently removed under mild basic conditions. Copyright

Pyridyl-alanine as a Hydrophilic, Aromatic Element in Peptide Structural Optimization

Glucagon (Gcg) 1 serves a seminal physiological role in buffering against hypoglycemia, but its poor biophysical properties severely complicate its medicinal use. We report a series of novel glucagon analogues of enhanced aqueous solubility and stability at neutral pH, anchored by Gcg[Aib16]. Incorporation of 3- and 4-pyridyl-alanine (3-Pal and 4-Pal) enhanced aqueous solubility of glucagon while maintaining biological properties. Relative to native hormone, analogue 9 (Gcg[3-Pal6,10,13, Aib16]) demonstrated superior biophysical character, better suitability for medicinal purposes, and comparable pharmacology against insulin-induced hypoglycemia in rats and pigs. Our data indicate that Pal is a versatile surrogate to natural aromatic amino acids and can be employed as an alternative or supplement with isoelectric adjustment to refine the biophysical character of peptide drug candidates.