Omniligase-1-Mediated Phage-Peptide Library Modification and Insulin Engineering.

Chemical and enzymatic modifications of peptide-displayed libraries have been successfully employed to expand the phage display library. However, the requirement of specific epitopes and scaffolds has limited the scope of protein engineering using phage display. In this study, we present a novel approach utilizing omniligase-1-mediated selective and specific ligation on the phage pIII protein, offering a high conversion rate and compatibility with commercially available phage libraries. We applied this method to perform high-throughput engineering of insulin analogues with randomized B chain C-terminal regions. Insulin analogues with different B chain C-terminal segments were selected and exhibited biological activity equivalent to that of human insulin. Molecular dynamics studies of insulin analogues revealed a novel interaction between the insulin B27 residue and insulin receptor L1 domain. In summary, our findings highlight the potential of omniligase-1-mediated phage display in the development and screening of disulfide-rich peptides and proteins. This approach holds promise for the creation of novel insulin analogues with enhanced therapeutic properties and exhibits potential for the development of other therapeutic compounds.

Suboptimal antimicrobial discharge prescriptions at a tertiary referral children's hospital.

Objective: To determine the rate of and factors associated with suboptimal discharge antimicrobial prescribing at a tertiary referral children's hospital. Design: Retrospective cohort. Setting: Tertiary referral children's hospital. Population: All enteral antimicrobial discharge prescriptions at Lucile Packard Children's Hospital Stanford from January 1st, 2021 through December 31st, 2021. Method: All enteral discharge antimicrobials are routinely evaluated by our antimicrobial stewardship program within 48 hours of hospital discharge. Antimicrobials are determined to be optimal or suboptimal by an antimicrobial stewardship pharmacist after evaluating the prescribed choice of antimicrobial, dose, duration, dosing frequency, and formulation. The rate and factors associated with suboptimal antimicrobial discharge prescribing were evaluated. Results: Of 2,593 antimicrobial prescriptions ordered at discharge, 19.7% were suboptimal. Suboptimal prescriptions were due to incorrect duration (72.2%), dose (31.0%), dose frequency (23.3%), drug choice (6.5%), or formulation (5.7%). In total, 87.2% of antimicrobials for perioperative prophylaxis and 13.5% of treatment antimicrobials were suboptimal. Antimicrobials with the highest rate of suboptimal prescriptions were amoxicillin-clavulanate (40.7%), clindamycin (36.6%), and cephalexin (36.6%). Conclusion: Suboptimal antimicrobial discharge prescriptions are common and present an opportunity for antimicrobial stewardship programs during hospital transition of care. Factors associated with suboptimal prescriptions differ by antimicrobial and prescribed indication, indicating that multiple stewardship interventions may be needed to improve prescribing.

Serine-mediated hydrazone ligation displaying insulin-like peptides on M13 phage pIII.

Phage display has emerged as a tool for the discovery of therapeutic antibodies and proteins. However, the effective display and engineering of structurally complex proteins, such as insulin, pose significant challenges due to the sequence of insulin, which is composed of two peptide chains linked by three disulfide bonds. In this study, we developed a new approach for the display of insulin-like peptides on M13 phage pIII, employing N-terminal serine-mediated hydrazone ligation. The insulin-displaying phage retains the biological binding affinity of human insulin. To address the viability loss after ligation, we introduced a trypsin-cleavable spacer on pIII, enabling insulin-displayed phage library selection. This method offers a general pathway for the display of structurally complex proteins on pIII, enhancing the practicality of selecting chemically modified phage libraries and opening avenues for the engineering of new insulin analogs for the treatment of diabetes by using phage display.

Supramolecular approaches for insulin stabilization without prolonged duration of action.

Aggregation represents a significant challenge for the long-term formulation stability of insulin therapeutics. The supramolecular PEGylation of insulin with conjugates of cucurbit[7]uril and polyethylene glycol (CB[7]‒PEG) has been shown to stabilize insulin formulations by reducing aggregation propensity. Yet prolonged in vivo duration of action, arising from sustained complex formation in the subcutaneous depot, limits the application scope for meal-time insulin uses and could increase hypoglycemic risk several hours after a meal. Supramolecular affinity of CB[7] in binding the B1-Phe residue on insulin is central to supramolecular PEGylation using this approach. Accordingly, here we synthesized N-terminal acid-modified insulin analogs to reduce CB[7] interaction affinity at physiological pH and reduce the duration of action by decreasing the subcutaneous depot effect of the formulation. These insulin analogs show weak to no interaction with CB[7]‒PEG at physiological pH but demonstrate high formulation stability at reduced pH. Accordingly, N-terminal modified analogs have in vitro and in vivo bioactivity comparable to native insulin. Furthermore, in a rat model of diabetes, the acid-modified insulin formulated with CB[7]‒PEG offers a reduced duration of action compared to native insulin formulated with CB[7]‒PEG. This work extends the application of supramolecular PEGylation of insulin to achieve enhanced stability while reducing the risks arising from a subcutaneous depot effect prolonging in vivo duration of action.

Synthesis and Characterization of Phenylboronic Acid-Modified Insulin With Glucose-Dependent Solubility.

Glucose-responsive insulin represents a promising approach to regulate blood glucose levels. We previously showed that attaching two fluorophenylboronic acid (FPBA) residues to the C-terminal B chain of insulin glargine led to glucose-dependent solubility. Herein, we demonstrated that relocating FPBA from B chain to A chain increased the baseline solubility without affecting its potency. Furthermore, increasing the number of FPBA groups led to increased glucose-dependent solubility.

Symmetric and asymmetric receptor conformation continuum induced by a new insulin.

Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.

Novel four-disulfide insulin analog with high aggregation stability and potency.

Although insulin was first purified and used therapeutically almost a century ago, there is still a need to improve therapeutic efficacy and patient convenience. A key challenge is the requirement for refrigeration to avoid inactivation of insulin by aggregation/fibrillation. Here, in an effort to mitigate this problem, we introduced a 4th disulfide bond between a C-terminal extended insulin A chain and residues near the C-terminus of the B chain. Insulin activity was retained by an analog with an additional disulfide bond between residues A22 and B22, while other linkages tested resulted in much reduced potency. Furthermore, the A22-B22 analog maintains the native insulin tertiary structure as demonstrated by X-ray crystal structure determination. We further demonstrate that this four-disulfide analog has similar in vivo potency in mice compared to native insulin and demonstrates higher aggregation stability. In conclusion, we have discovered a novel four-disulfide insulin analog with high aggregation stability and potency.