Design and Biological Evaluation of the Long-Acting C5-Inhibited Ornithodoros moubata Complement Inhibitor (OmCI) Modified with Fatty Acid.

Disorder of complement response is a significant pathogenic factor causing some autoimmune and inflammation diseases. The Ornithodoros moubata Complement Inhibitor (OmCI), a small 17 kDa natural protein, was initially extracted from soft tick salivary glands. The protein was found binding to complement C5 specifically, inhibiting the activation of the complement pathway, which is a successful therapeutic basis of complement-mediated diseases. However, a short half-life due to rapid renal clearance is a common limitation of small proteins for clinical application. In this study, we extended the half-life of OmCI by modifying it with fatty acid, which was a method used to improve the pharmacokinetics of native peptides and proteins. Five OmCI mutants were initially designed, and single-site cysteine mutation was introduced to each of them. After purification, four OmCI mutants were obtained that showed similar in vitro biological activities. Three mutants of them were subsequently coupled with different fatty acids by nucleophilic substitution. In total, 15 modified derivatives were screened and tested for anticomplement activity in vitro. The results showed that coupling with fatty acid would not significantly affect their complement-inhibitory activity (CH50 and AH50). OmCIT90C-CM02 and OmCIT90C-CM05 were validated as the applicable OmCI bioconjugates for further pharmacokinetic assessments, and both showed improved plasma half-life in mice compared with unmodified OmCI (15.86, 17.96 vs 2.57 h). In summary, our data demonstrated that OmCI conjugated with fatty acid could be developed as the potential long-acting C5 complement inhibitor in the clinic.

Design of a novel long-acting dual GLP-1/GIP receptor agonist.

Tirzepatide, the first approved dual GLP-1/GIP receptor agonist (RA), has achieved better clinical outcomes than other GLP-1RAs. However, it is an imbalanced dual GIP/GLP-1 RA, and it remains unclear whether the degree of imbalance is optimal. Here, we present a novel long-acting dual GLP-1/GIP RA that exhibits better activity than tirzepatide toward GLP-1R. A candidate conjugate, D314, identified via peptide design, synthesis, conjugation, and experimentation, was evaluated using chronic studies in db/db and diet induced obese (DIO) mice. D314 achieved favorable blood glucose and body weight-lowering effects, equal to those of tirzepatide. Its half-life in dogs (T1/2: 78.3 ± 14.01 h) reveals its suitability for once-weekly administration in humans. This preclinical study suggests the potential role of D314 as an effective agent for treating T2DM and obesity.

Design, synthesis, and biological evaluation of a potential long-acting glucagon-like peptide-1 (GLP-1) analog.

By binding to its receptor, glucagon-like peptide-1 (GLP-1) plays various physiological roles, including activating glucose-dependent insulin secretion, inhibiting gastric emptying, and reducing appetite. This suite of activities makes GLP-1 and its analogs an attractive choice for treating type 2 diabetes mellitus in the context of overweight or obesity. This study used different types and lengths of fatty acids to design dual fatty acid side chains for GLP-1 receptor agonists including decanoic, dodecanoic, tetradecanoic, hexadecanoic, dodecanedioic, tetradecanedioic, hexadecanedioic, and octadecanedioic acids. Sixteen GLP-1 receptor agonists (conjugates 13-28) with dual fatty acid side chains were obtained by liquid-phase synthesis. After structural confirmation using high-resolution mass spectrometry, peptide mapping, and circular dichroism, the biological activities of the conjugates were screened. First, the conjugates were screened for albumin binding and activity in GLP-1R-CRE-bla CHO-K1 cells. Albumin binding results suggested a synergistic effect between the two fatty acids in the conjugates. Next, conjugates 18, 19, and 21 selected after primary screening were assessed for receptor affinity, activity in INS-1 cells, plasma stability across different species, and efficacy and pharmacokinetics in normal and db/db mice. One candidate (conjugate 19) was found to have albumin binding of >99 %, good receptor affinity, activities of INS-1 cells, and plasma stability. We found that cellular activities in GLP-1R-CRE-bla CHO-K1 cells and pharmacodynamics and pharmacokinetics in normal and db/db mice for conjugate 19 were superior to those of semaglutide.

Design, synthesis and antibacterial activity of novel colistin derivatives with thioether bond-mediated cyclic scaffold.

The escalating crisis of multidrug resistance is raising the fear of untreatable Gram-negative infections and killing a substantial number of patients. The underpopulated antibiotic drug development pipelines drive polymyxins (polymyxin B and colistin) as crucial therapeutic options. However, the cumbersome synthesis process and inefficient cyclization method limit the efficient preparation of polymyxin core scaffolds in the development of polymyxin derivatives. Here, we innovatively applied a substitution reaction between bromobenzene and sulfhydryl to cyclize colistin core scaffolds. The reaction was mild and efficient, improving the total yield of the compound from less than 10% to 55.90%. Nearly 30 novel derivatives with thioether bond-mediated cyclic scaffolds were designed and synthesized. Evaluation of antibacterial activities and biological properties revealed that many new compounds that are stable in mouse plasma possess high antimicrobial potency against Gram-negative bacteria and display no hemolytic toxicity. Our optimal peptide PE-2C-C8-DH eradicated Acinetobacter baumannii within 24 h in vitro, and had lower acute toxicity and significant therapeutic effects on mice infected with Pseudomonas aeruginosa, which deserves further development.

Hectogram-scale synthesis of [Aib8 , Arg34 ]-GLP-1 (7-37) by liquid-phase fragment condensation.

Based on small-scale synthesis (0.3 g), a 100-g scale-up synthesis of crude [Aib8 , Arg34 ]-glucagon-like peptide-1 (GLP-1) (7-37) was completed. The crude [Aib8 , Arg34 ]-GLP-1 (7-37) was purified using a dynamic axial compression column 200 (DAC-200). Approximately 61 g of [Aib8 , Arg34 ]-GLP-1 (7-37) with a purity of >99% was obtained through one-step reverse-phase chromatography. The purification yield was approximately 92%. The yield from the total reaction was approximately 60%. In summary, we developed an economical and environmentally friendly route to the synthesis and purification of crude [Aib8 , Arg34 ]-GLP-1 (7-37), laying a foundation for subsequent industrial production.

Synthesis and biological evaluation of glucagon-like peptide-1 analogs with the C-terminal helix 3 of albumin-binding domain 3.

Based on peptide 6 (Ser8-GLP-1 [7-35]-GVKALIDEILAA-NH2; GVKALI-DEILAA is the C-terminal helix 3 of albumin-binding domain 3 of protein G from bacterial Streptococcal G strain 148 [G148-ABD3]), a series of its analogs (compounds 0-VI: Aib8-GLP-1 [7-35]-linkers-GVKALIDEILAA-NH2) were designed and synthesized using microwave-assisted solid-phase synthesis. First, to monitor the reaction process and reduce potential risks, the synthesis process of compounds 0-VI was divided into three stages. Next, to explore the effect of these linkers on their albumin-binding rates, albumin-binding assays were performed. Finally, to evaluate their biological activities in vitro and in vivo, receptor potency, surface plasmon resonance (SPR), weight-loss, and glucose-lowering assays were carried out. These results indicated the linkers of different polarities between Aib8-GLP-1 (7-35) and the C-terminal helix 3 of ABD3 can significantly affect the albumin-binding rate of the C-terminal helix 3 of ABD3. And compound IV had the highest albumin-binding rates, weight-loss, and glucose-lowering effects among them.

Efficient synthesis of Aib8 -Arg34 -GLP-1 (7-37) by liquid-phase fragment condensation.

Glucagon-like peptide-1 (GLP-1) is a potential therapeutic agent for treating Type 2 diabetes, owing to its glucose-dependent capability to stimulate insulin secretion. Semaglutide is currently the best GLP-1 analogue; however, the Aib8 -Arg34 -GLP-1 (7-37) of semaglutide contains an unnatural amino acid at the eighth position (Aib: 2-aminoisobutyric acid), which hinders its fermentation process. Aib8 -Arg34 -GLP-1 (7-37) is mainly synthesised by solid-phase synthesis. However, solid-phase synthesis of Aib8 -Arg34 -GLP-1 (7-37) has many shortcomings: (i) The synthesis requires many organic solvents, (ii) the existence of deletion peptides impedes the subsequent purification process, (iii) the yield is low (approximately 16%), and (iv) it is not suitable for large-scale synthesis. However, the synthesis of Aib8 -Arg34 -GLP-1 (7-37) by liquid-phase fragment condensation of expressed and synthetic peptides (Arg34 -GLP-1 (9-37) and Boc-His (Boc)-Aib) has many advantages: (i) The synthesis process only requires a few organic reagents, (ii) the yield is high (approximately 60%), (iii) the purification conditions are simple and Aib8 -Arg34 -GLP-1 (7-37) with a purity of over 98% is obtained through one-step reverse-phase purification, and (iv) the raw materials are inexpensive and large-scale synthesis is possible. In conclusion, here, we developed an efficient method for synthesising Aib8 -Arg34 -GLP-1 (7-37).

Discovery of potent and selective Bcl-2 inhibitors with acyl sulfonamide skeleton.

The antiapoptotic protein B-cell lymphoma 2 (Bcl-2), overexpressed in many tumor cells, is an attractive target for potential small molecule anticancer drug discovery. Herein, a series of novel derivatives with acyl sulfonamide skeleton was designed, synthesized, and evaluated as Bcl-2 inhibitors by means of bioisosteric replacement. Among them, compound 24g demonstrated equal efficient inhibition activity against RS4;11 cell line compared to positive control ABT-199. Moreover, it showed improved selectivity for Bcl-2/Bcl-xL inhibitory effects, the result of which was consistent with platelet toxicity studies. In vitro and in vivo pharmacokinetic properties of compound 24g had a significantly improved profiles. Taken together, those results suggested it as a promising candidate for development of novel therapeutics targeting Bcl-2 in cancer.

Discovery of a Long-Acting Parathyroid Hormone 1-34 Analogue to Treat Hypoparathyroidism.

Hypoparathyroidism (HP) is a rare disease with clinical manifestations of hypocalcemia and hyperphosphatemia, resulting from deficient or absent parathyroid hormone (PTH) secretion. Conventional treatment for patients with HP involves extensive calcium and vitamin D supplementation. In 2015, PTH1-84 was approved by the United States Food and Drug Administration as an adjunct for HP patients who cannot be well-controlled on conventional treatment. However, PTH1-84 therapy requires a daily injection, leading to poor patient compliance. The purpose of this study was to develop a long-acting PTH1-34 analogue by increasing its affinity to albumin. Three PTH1-34 variants were generated by substituting two of the three lysine (Lys) residues with arginine, reserving a single Lys as the modification site in each sequence. A series of side chains, containing fatty acid, deoxycholic acid, or biotin groups, were synthesized to modify these PTH1-34 variants by using a solid-liquid phase synthesis approach. In vitro bioactivity and albumin affinity tests were used to screen these new PTH1-34 analogues. Finally, Lys27-AAPC was selected from 69 synthesized analogues as a candidate therapeutic compound because it retained potency and exhibited a high albumin-binding capacity. In pharmacodynamic experiments, Lys27-AAPC demonstrated enhanced and prolonged efficacy in serum calcium elevating relative to PTH1-84. Moreover, a lyophilized powder for injection containing Lys27-AAPC was developed for further testing and represented a potential long-acting HP treatment.

Design, synthesis and biological evaluation of double fatty chain-modified glucagon-like peptide-1 conjugates.

Twelve double fatty chains and Aib8-Arg34-GLP-1 (7-37) were designed and obtained by microwave-assisted solid-phase synthesis. Then, twelve conjugates of Aib8-Arg34-GLP-1 (7-37) were synthesized in 1% triethylamine aqueous solution. Conjugates 2, 3, 6, 7, 10 and 11 showed better GLP-1 receptor activation potency than semaglutide. However, conjugates 2, 6 and 10 showed slightly worse glucose-lowering effects in vivo than semaglutide but better effects than conjugates 3, 7 and 11. The CD spectra of conjugates 2, 6 and 10 indicated that they had the same secondary structure as liraglutide and semaglutide. The receptor affinity results for conjugates 2, 6 and 10 measured by SPR (surface plasmon resonance) showed that conjugate 2 had higher receptor affinity than conjugates 6 and 10. In addition, albumin binding assays indicated that double fatty acid chains had obvious synergistic effects compared with single fatty acid chains. In conclusion, the structure-activity relationship of different side chains was summarized and one candidate, conjugate 2, was screened.

Synthesis and biological evaluation of esterified and acylated derivatives of NH2-(AEEA)5-amphotericin B.

Based on NH2-(AEEA)5-amphotericin B (DMR005; AEEA is 8-amino-3,6-dioxaoctanoic acid), a series of novel esterified and acylated derivatives of DMR005 were synthesized. These derivatives were evaluated for their antifungal activities using the broth dilution method, for their hemolytic toxicity with sterile defibrinated sheep blood, and for their self-association through UV-visible spectroscopy. The preliminary screening tests indicated that NH2-(AEEA)5-amphotericin B methyl ester (DMR031) was an ideal compound. The results of minimum inhibitory concentration and time-kill assays showed that antifungal activities of DMR031 (4 µg ml-1) against Candida albicans ATCC10231 and ATCC90028 were reduced by four times compared to these of amphotericin B (AmB) (1 µg ml-1). DMR031 (142 ± 1 mg ml-1) significantly improved the water solubility of AmB as DMR005 did. Preliminary safety assessments of DMR031 were carried out via cell toxicity assay of HEK293T in vitro, which turned out to be much better than AmB. AmB had good efficacy in vivo at a dose of 1 mg ml-1. However, DMR031 still had no efficacy in vivo even at a dose of 16 mg ml-1, merely prolonged the survival time of mice.

The synthesis and activity evaluation of N-acylated analogs of echinocandin B with improved solubility and lower toxicity.

Presently, echinocandins have been recommended as the first-line drugs for the treatment of invasive candidiasis. However, low oral bioavailability and solubility limit their application. To improve this situation, this study chose amino acid and fatty acid as raw materials to modify the nucleus of echinocandin B. Six N-acylated analogs were screened from the derivatives that possessed potent antifungal activity and good water solubility. Based on antifungal susceptibility and hemolytic toxicity, compound 5 as the candidate had good antifungal activity and no hemolytic effect. Moreover, compared with anidulafungin, compound 5 showed a comparable fungicidal effect, much higher solubility, and lower toxicity. In conclusion, compound 5 has the potential for further research and development on account of reserved antifungal activity, high solubility, and low toxicity.

Design, synthesis and antimicrobial studies of some polymyxin analogues.

To find novel polymycin analogues with high antimicrobial activities and low toxicity, 36 novel polymyxin analogues were synthesized, and in which TZ40-J and TZ40-K were evaluated for their antimicrobial activities using broth microdilution method and for their haemolytic toxicity with sterile sheep blood. Preliminary safety assessments of those two compounds were carried out via the MTT cell viability assay in vitro and acute toxicity assay in vivo. Experimental data demonstrated that TZ40-J and TZ40-K were less toxic and indicate higher activities against Pseudomonas aeruginosa than polymyxin B.

Recognition of Invasive Prostate Cancer Using a GHRL Polypeptide Probe Targeting GHSR in a Mouse Model In Vivo.

BACKGROUND: Ghrelin (GHRL) is a polypeptide that can specifically bind to the growth hormone secretagogue receptor (GHSR). The expression of GHSR is significantly different in normal and prostate cancer (PC) tissues in humans. It is important to find an effective diagnostic method for the diagnosis and prognosis of invasive PC/neuroendocrine prostate cancer (NEPC). METHODS: GHRL and GHSR mRNA levels were determined by a quantitative real-time polymerase chain reaction in PC tissues. The expression of GHRL and GHSR proteins was assessed by Western blot assay and immunohistochemistry. A GHRL polypeptide probe was synthesized by standard solid-phase polypeptide synthesis, and labeled with Alexa Fluor 660. Confocal microscopy was used to capture fluorescence images. Living imaging analysis showed tumor areas of different invasiveness in mice models. RESULTS: The levels of GHRL and GHSR copy number amplification and mRNA expression were increased in invasive PC/NEPC, and the protein expression levels of GHRL and GHSR were similarly increased in NEPC. The GHRL polypeptide probe could effectively bind to GHSR. In PC3 cells, it was found that the GHRL probe specifically binds to GHSR on the cell membrane and accumulates in the cells through internalization after binding. Live imaging in mice models showed that there were different signal intensities in tumor areas with different invasiveness. CONCLUSION: GHSR and GHRL might be used in molecular imaging diagnosis for invasive PC/NEPC in the future.

Synthesis and characterization of NH2-(AEEA)n-amphotericin B derivatives.

To find novel amphotericin B (AmB) derivatives with high therapeutic potential, low toxicity, and water solubility, a series of nine N-substituted AmB derivatives were evaluated for their antifungal activity using the broth dilution method and for their hemolytic toxicity with sterile defibrinated sheep blood. Qualitative screening of the effect of the derivatives on two reference Candida albicans strains and of their solubility was performed based on the value of n (n is a positive integer), resulting in the identification of an optimal compound, NH2-(AEEA)5-AmB (DMR005; AEEA is 8-amino-3,6- dioxaoctanoic acid). Preliminary safety assessments of DMR005 were carried out via the MTT cell viability assay in vitro and acute toxicity assay in vivo. In general, DMR005 not only has higher water solubility and less toxicity than the parent polyene but also retains antifungal potency.