David B. Flora

Modifications in the B10 and B26–30 regions of the B chain of human insulin alter affinity for the human IGF-I receptor more than for the insulin receptor

Summary Inversion of the natural sequence of the B chain of human insulin (HI) from ProB28LysB29 to LysB28ProB29 generates an insulin analogue with reduced tendency to self-associate. Since this substitution increases the homology of insulin to insulin-like growth factor-I (IGF-I), we have examined the affinity of a series of insulin analogues with the general modified structure XaaB28ProB29 HI for binding to both human placental insulin and IGF-I receptors. The XaaB28ProB29 HI series is approximately equipotent to HI in binding to the insulin receptor with the exception of when Xaa = Phe, Trp, Leu, Ile, and Gly (40–60 % relative to HI). Substitution with basic residues in the B28 position increased the relative affinity to the IGF-I receptor approximately 1.5−2-fold (ArgB28ProB29 > OrnB28ProB29 = LysB28ProB29). Substitution with acidic residues reduced relative affinity for the IGF-I receptor approximately 2-fold (CyaB28ProB29 = GluB28ProB29 > AspB28ProB29). Combination of AspB10 substitution in conjunction with a modification in the B28–29 position (e.g. AspB10LysB28ProB29 HI) showed an additional 2-fold selective increase in affinity for the IGF-I receptor, suggesting that these two effects are additive. Addition of Arg residues at B31–32, on the backbone of either HI or AspB10 HI, increased affinity for the IGF-I receptor 10 and 28 fold, respectively, compared to HI, confirming the significance of enhanced positive charge at the C-terminal end of the insulin B-chain in increasing selectivity for the IGF-I receptor. This relative increase in IGF-I receptor affinity correlated largely, but not completely, with enhanced growth promoting activity in human mammary epithelial cells. In the case of LysB28ProB29 HI, growth activity correlated with dissociation kinetics from the insulin receptor which were shown to be identical with those of human insulin. [Diabetologia (1997) 40: S 54–S 61]

Hybrid insulin cocrystals for controlled release delivery

The ability to tailor the release profile of a drug by manipulating its formulation matrix offers important therapeutic advantages. We show here that human insulin can be cocrystallized at preselected ratios with the fully active lipophilically modified insulin derivative octanoyl-Nε-LysB29–human insulin (C8-HI). The cocrystal is analogous to the NPH (neutral protamine Hagedorn) crystalline complex formed with human insulin, which is commonly used as the long-acting insulin component of diabetes therapy. The in vitro and in vivo release rates of the cocrystal can be controlled by adjusting the relative proportions of the two insulin components. We identified a cocrystal composition comprising 75% C8-HI and 25% human insulin that exhibits near-ideal basal pharmacodynamics in somatostatin-treated beagle dogs. The dependence of release rate on cocrystal ratio provides a robust mechanism for modulating insulin pharmacodynamics. These findings show that a crystalline protein matrix may accommodate a chemical modification that alters the dissolution rate of the crystal in a therapeutically useful way, yet that is structurally innocuous enough to preserve the pharmaceutical integrity of the original microcrystalline entity and the pharmacological activity of the parent molecule.

Self-Association Properties of Monomeric Insulin Analogs Under Formulation Conditions

AbstractPurpose. The purpose of the current study was to investigate the effects of two important excipients, zinc and m-cresol, on the self-association properties of a series of monomeric insulin analogs. In this way, the effects on formulation behavior of individual amino acid substitutions in the C-terminal region of the insulin B-chain could be compared. Methods. The self-association of ten insulin analogs was monitored by equilibrium and velocity analytical ultracentrifugation under three different conditions: (i) in neutral buffer alone; (ii) in neutral buffer containing zinc ion; and (iii) in neutral buffer containing both zinc ion and phenolic preservative (a typical condition for insulin formulations). The self-association properties of these analogs were compared to those of human insulin and the rapid-acting insulin analog LysB28ProB29-human insulin. Results. The analogs in the current study exhibited a wide range of association properties when examined in neutral buffer alone or in neutral buffer containing zinc ion. However, all of these analogs had association properties similar to human insulin in the presence of both zinc and m-cresol. Under these formulation conditions each analog had an apparent sedimentation coefficient of s* = 2.9−3.1 S, which corresponds to the insulin hexamer. Conclusions. Analogs with changes in the B27−B29 region of human insulin form soluble hexamers in the presence of both zinc and m-cresol, and m-cresol binding overrides the otherwise destabilizing effects of these mutations on self assembly.

A Synthetic Route to Human Insulin Using Isoacyl Peptides

The chemical synthesis of insulin has been a longstanding challenge, mainly because of the notorious hydrophobicity of the A chain and the complicated topology of this 51-mer peptide hormone consisting of two chains and three disulfide bonds. Reported herein is a new synthetic route utilizing the isoacyl peptide approach to address the hydrophobicity problems. The incorporation of isoacyl dipeptide segments into both A and B chains greatly improved their preparation and purification, and the RP-HPLC recovery of the chain ligation intermediates. The new route affords human insulin with a yield of 68 % based on the starting purified A chain and an overall yield of 24 % based on the substitution of the resin used for the preparation of A chain. To the best of our knowledge, this represents the most efficient route of human insulin chemical synthesis reported to date.

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.

Structure-Activity Relationships of β -MSH Derived Melanocortin-4 Receptor Peptide Agonists

The recent emergence of obesity as a major health threat in the industrialized world has intensified the search for novel and effective pharmacologic treatment. The proopiomelanocortin (POMC)-melanocortin 4 receptor (MC4R) axis has been shown to regulate food intake and energy homeostasis and is considered among the most promising antiobesity targets. Our initial efforts in this area have focused on affinity and selectivity directed optimization of the native beta-MSH(5-22) sequence and resulted in the discovery of a potent MC4R agonist: Ac-Tyr-Arg-[Cys-Glu-His-D-Phe-Arg-Trp-Cys]-NH(2) (10). Subcutaneous administration of this peptide produced an excellent in vivo efficacy in reducing food intake and increasing fat metabolism. Additionally, suppression of food intake was observed in wild type but not in MC4R deficient mice, suggesting that the effects observed in the wild type mice were mediated through MC4R signaling. Subsequent optimization efforts led to the identification of a novel series of disulfide constrained hexapeptides as exemplified by Ac-[hCys-His-D-Phe-Arg-Trp-Cys]-NH(2) (100). These cyclic hexapeptides showed a further improved potency in binding MC4R and an enhanced selectivity over MC1R. At a dose of 0.07 mg/kg analog 102 reduced food intake by 38% and increased fat utilization by 58% in rats. These cyclic peptides provide novel and enhanced reagents for the elucidation of melanocortin receptors biology and may find applications in the treatment of obesity and related metabolic disorders.

Discovery of potent, cyclic calcitonin gene-related peptide receptor antagonists.

Calcitonin gene‐related peptide (CGRP), a potent dilator of cerebral and dural vasculature, is known to be elevated in plasma and cerebral spinal fluid during migraine attacks. Selective blockade of the CGRP receptor offers the promise of controlling migraine headache more effectively and without the side‐effects associated with the use of triptans. Our efforts to develop a novel, peptide‐based CGRP antagonist focused on the C‐terminal portion of the peptide which is known to bind the receptor but lack agonist properties. Extensive SAR studies of the C‐terminal CGRP (27–37) region identified a novel cyclic structure: Bz‐Val‐Tyr‐cyclo[Cys‐Thr‐Asp‐Val‐Gly‐Pro‐Phe‐Cys]‐Phe‐NH2 (23) with a kb value of 0.126 nM against the cloned human CGRP receptor. Additional SAR studies directed at enhancement of potency and improvement of physicochemical properties yielded a series of analogs with kb values in the 0.05–0.10 nM range. Copyright

Optimization of the Chemical Synthesis of Human Ghrelin

Since its initial discovery and characterization as the native growth hormone secretagogue [1], ghrelin GSS(n-octanoyl)FLSPEHQRVQQRKESKKPPAKLQPR has stimulated extensive studies directed toward clarifying its role in feeding, energy balance and growth hormone release [2–5]. A flexible and efficient synthetic procedure was required to produce sufficient quantities of human ghrelin in order to conduct structure-activity studies related to the native peptide. To this end we carried out four separate solid-phase Fmoc/tBu based synthetic protocols which differed with respect to the method of incorporation of the Ser3-octanoyl moiety. The methods were evaluated for yield and purity of final product. In addition, we optimized a procedure for the esterification of the Ser3 side chain with respect to stoichiometry and time course.