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.

Long-Term Strategies for Poorly Water-Soluble Peptides: Combining Fatty Acid Modification with PAS Fusion.

Bulevirtide, an entry inhibitor for the hepatitis B virus (HBV) and hepatitis D virus (HDV), is currently available on the European market. However, its clinical application is constrained by its short half-life and poor water solubility, rendering it unsuitable for fatty acid modification, aimed at achieving long-term effects. To address this limitation, we integrated a polypeptide chain consisting of Pro, Ala, and Ser at the C-terminus, which increased its hydrophilicity. To obtain the fusion sequence of A1 and A2, encompassing amino acids 1-47 of Bulevirtide and PAS, we used Escherichia coli fermentation expression. Subsequently, the N-terminal myristoyl groups of A1 and A2 were modified to yield Myr-A1 and Myr-A2, respectively. Five fatty acid moieties with the same hydrophilic spacers and different fatty acids were conjugated to analogs, generating 10 bioconjugations. The bioconjugates were then evaluated for their anti-HBV activity. Among them, HB-10 was selected for pharmacokinetic analysis and demonstrated a significantly prolonged half-life, with 5.88- and 13.18-fold increases in beagle dogs and rats, respectively. Additionally, higher drug doses resulted in substantially elevated liver concentrations. In conclusion, via fatty acid incorporation and PASylation, we successfully developed a novel Bulevirtide bioconjugate, HB-10, that exhibits an extended action duration. This compound holds substantial promise as a prospective long-acting entry inhibitor, warranting further investigation.

Discovery of imidazo[1,2-b]pyridazine macrocyclic derivatives as novel ALK inhibitors capable of combating multiple resistant mutants.

Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) often loses effectiveness against non-small cell lung malignancies (NSCLCs) with ALK gene rearrangements (ALK+). 19 novel imidazo[1,2-b]pyridazine macrocyclic derivatives were designed, synthesized, and tested for their biological activities in an effort to develop ALK inhibitors that would overcome second-generation ALK-TKIs, particularly the G1202R mutation and the lorlatinib-resistant L1196M/G1202R double mutations. Of all the target substances, O-10 had the most effective enzymatic inhibitory activity, with IC50 values for ALKWT, ALKG1202R, and ALKL1196M/G1202R of 2.6, 6.4, and 23 nM, respectively. O-10, on the other hand, reduced the growth of ALK-positive Karpas299, BaF3-EML4-ALKG1202R, and BaF3-EML4-ALKL1196M/G1202R cells with IC50 values of 38, 52, and 64 nM, respectively. This was equally effective to the reference drug Repotrectinib (IC50 = 40, 164, and 208 nM). The kinase selectivity profile, liver microsome stability test and in vivo pharmacokinetic properties in SD rats of compound O-10 were further evaluated. O-10 was regarded as an effective ALK inhibitor for the treatment of mutations overall.

Design, synthesis and biological evaluation of novel N-(methyl-d3) pyridazine-3-carboxamide derivatives as TYK2 inhibitors.

As a mediator of pro-inflammatory cytokines, TYK2 is an attractive target to treat autoimmunity diseases. Herein, we reported the design, synthesis, and structure-activity relationships (SARs) of N-(methyl-d3) pyridazine-3-carboxamide derivatives as TYK2 inhibitors. Among them, compound 24 exhibited acceptable inhibition activity against STAT3 phosphorylation. Furthermore, 24 showed satisfactory selectivities toward other members of JAK family and performed a good stability profile in liver microsomal assay. Pharmacokinetics (PK) study indicated that compound 24 has reasonable PK exposures. In anti-CD40-induced colitis models, compound 24 was orally highly effective with no significant hERG and CYP isozymes inhibition. These results indicated that compound 24 was worthy of further investigation for the development of anti-autoimmunity diseases agents.

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 and synthesis of boron-containing ALK inhibitor with favorable in vivo efficacy.

ALK is an attractive therapeutic target for the treatment of non-small cell lung cancer. As an emerging element in medicinal chemistry, boron has achieved great success in the discovery of antitumor drugs and antibacterial agents. Through construction of a BCC (boron-containing compound) compound library and broad kinase screening, we found the ALK inhibitor hit compound 10a. Structural optimization by CADD and isosterism revealed that lead compound 10k has improved activity (ALKL1196M IC50 = 8.4 nM, NCI-H2228 cells IC50 = 520 nM) and better in vitro metabolic stability (human liver microsomes, T1/2 = 238 min). Compound 10k showed good in vivo efficacy in a nude mouse NCI-H2228 lung cancer xenograft model with a TGI of 52 %. Molecular simulation analysis results show that the hydroxyl group on the oxaborole forms a key hydrogen bond with Asn1254 or Asp1270, and this binding site provides a new idea for drug design. This is the first publicly reported lead compound for a boron-containing ALK inhibitor.

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.

Design and synthesis of boron-containing diphenylpyrimidines as potent BTK and JAK3 dual inhibitors.

Bruton's tyrosine kinase (BTK) and Janus kinase 3 (JAK3) are very promising targets for hematological malignancies and autoimmune diseases. In recent years, a few compounds have been approved as a marketed medicine, and several are undergoing clinical trials. By recombining the dominant backbone of known active compounds, constructing a foused library, and screening a broad panel of kinases, we found a class of compounds with dual activities of anti-BTK and anti-JAK3. Some of the compounds have shown 10-folds more active in the enzyme and cell-based assays than a known active compound. Furthermore, liver microsome stability experiments show that these compounds have better stability than ibrutinib. These explorations offered new clues to discover benzoxaborole fragment and pyrimidine scaffold as more effective BTK and JAK3 dual inhibitors.

Magnesium isoglycyrrhizinate ameliorates high fructose-induced liver fibrosis in rat by increasing miR-375-3p to suppress JAK2/STAT3 pathway and TGF-ß1/Smad signaling.

Increasing evidence has demonstrated that excessive fructose intake induces liver fibrosis. Epithelial-mesenchymal transition (EMT) driven by transforming growth factor-ß1 (TGF-ß1)/mothers against decapentaplegic homolog (Smad) signaling activation promotes the occurrence and development of liver fibrosis. Magnesium isoglycyrrhizinate is clinically used as a hepatoprotective agent to treat liver fibrosis, but its underlying molecular mechanism has not been identified. Using a rat model, we found that high fructose intake reduced microRNA (miR)-375-3p expression and activated the janus-activating kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) cascade and TGF-ß1/Smad signaling, which is consistent with the EMT and liver fibrosis. To further verify these observations, BRL-3A cells and/or primary rat hepatocytes were exposed to high fructose and/or transfected with a miR-375-3p mimic or inhibitor or treated with a JAK2 inhibitor, and we found that the low expression of miR-375-3p could induce the JAK2/STAT3 pathway to activate TGF-ß1/Smad signaling and promote the EMT. Magnesium isoglycyrrhizinate was found to ameliorate high fructose-induced EMT and liver fibrosis in rats. More importantly, magnesium isoglycyrrhizinate increased miR-375-3p expression to suppress the JAK2/STAT3 pathway and TGF-ß1/Smad signaling in these animal and cell models. This study provides evidence showing that magnesium isoglycyrrhizinate attenuates liver fibrosis associated with a high fructose diet.

Synthesis and biological evaluation of deuterated sofosbuvir analogs as HCV NS5B inhibitors with enhanced pharmacokinetic properties.

A series of deuterated sofosbuvir analogs were designed and prepared with the aim of improving their pharmacokinetic properties. The devised synthetic routes allow for site-selective deuterium incorporation with high levels of isotopic purity. As expected, the deuterated analogs (37-44) are as efficacious as sofosbuvir when tested in vitro inhibition of viral replication (replicon) assays. Compared with sofosbuvir, deuterated analog 40 displays improved in vivo pharmacokinetics profiles in rats and dogs in terms of the metabolite and the prodrug. The Cmax and area under the curve (AUC) of 40 in dogs were increased by 3.4- and 2.7-fold, respectively. Due to the enhanced pharmacokinetic properties and the great synthetic advantage of an inexpensive deuterium source (D2 O) for 40, it was chosen for further investigation.

Structure-activity relationship study of a series of caspase inhibitors containing γ-amino acid moiety for treatment of cholestatic liver disease.

A series of caspase inhibitors containing γ-amino acid moiety have been synthesized. A systemic study on their structure-activity relationship of anti-apoptotic cellular activity is presented. These efforts led to the discovery of compound 20o as a potent caspase inhibitor, which demonstrated preclinical ameliorating total bilirubin efficacy with a significantly improved pharmacokinetic profile.

C-Aryl glucoside SGLT2 inhibitors containing a biphenyl motif as potential anti-diabetic agents.

A series of highly active C-aryl glucoside SGLT2 inhibitors containing a biphenyl motif were designed and synthesized for biological evaluation. Among the compounds tested, compound 16l demonstrated high inhibitory activity against SGLT2 (IC50=1.9 nM) with an excellent pharmacokinetic profile. Further study indicated that the in vivo efficacy of compound 16l was comparable to that of dapagliflozin, suggesting that further development would be worthwhile.

Discovery of potent dipeptidyl peptidase IV inhibitors through pharmacophore hybridization and hit-to-lead optimization.

A novel dipeptidyl peptidase IV inhibitor hit (5, IC50=0.86 µM) was structurally derived from our recently disclosed preclinical candidate 4 by replacing the cyanobenzyl with a butynyl based on pharmacophore hybridization. A hit-to-lead optimization effort was then initiated to improve its potency. Most N-substituted analogs exhibited good in vitro activity, and compound 18o (IC50=1.55 nM) was identified to be a potent dipeptidyl peptidase IV inhibitor with a significantly improved pharmacokinetic properties (bioavailablity: 41% vs 82.9%; T1/2: 2h vs 4.9h).

Discovery, optimization and biological evaluation of novel HBsAg production inhibitors.

Hepatitis B virus (HBV) infection is a major global health problem. HBsAg inhibitors are expected to reduce the production of HBsAg via inhibiting host proteins PAPD5 & PAPD7 and finally achieve the ideal goal of "functional cure". In this work, a series of tetrahydropyridine (THP) derivatives with a bridged ring were synthesized and evaluated for their inhibiting HBsAg production and HBV DNA activity. Among them, compound 17i was identified as potent HBsAg production inhibitor with excellent in vitro anti-HBV potency (HBV DNA EC50 = 0.018 µM, HBsAg EC50 = 0.044 µM) and low toxicity (CC50 > 100 µM). Moreover, 17i exhibited favorable in vitro/in vivo DMPK properties in mice. 17i could also significantly reduce serum HBsAg and HBV DNA levels (1.08 and 1.04 log units, respectively) in HBV transgenic mice.

A quantitative method for determination of colistin E2 methanesulphonate in human plasma by 15N-labeled colistin E2.

The single-component colistin E2, with superior antibacterial activity and lower toxicity, was being developed as the latest generation of polymyxin drugs. However, colistin E2 has not been tested quantitatively in biological matrices. In this study, based on the quantitative detection of colistin methanesulphonate (CMS) and colistin by Zhao et al., 15N-labeled colistin E2 was used as an internal standard (IS) for a more accurate quantitative detection of CMS E2 in human plasma. A rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay method was developed for determination of CMS E2 and colistin E2 in human plasma. After pretreatment of plasma samples by 96-well SPE Supra-Clean Weak Cation Exchange (WCX) plate, the formed colistin E2 was detected and quantified by UHPLC-MS/MS system. All plasma lots were found to be free of interferences with the analyte. The matrix has no effect on the quantitation of the analyte. No significant effect of the carryover was observed. The dilution integrity was demonstrated in plasma samples without the loss of accuracy and precision. The lower limit of quantification (LLOQ) was 0.0300 mg/L for colistin E2 in plasma with accuracy (relative error, 5.1-12.7%) and precision (relative standard deviation, - 5.7-9.3%). Stability of CMS E2 and colistin E2 was demonstrated in biological samples before and during sample treatment, and in the extract. Furthermore, this method was successfully applied to the analysis of plasma samples obtained from Chinese healthy volunteers receiving a single intravenous CMS E2 dose of 5 mg/kg. In conclusion, the detection method was characterized by speed and high accuracy, which laid a solid foundation for the subsequent development of CMS E2 drug.

Novel TYK2 Inhibitors with an N-(Methyl-d 3)pyridazine-3-carboxamide Skeleton for the Treatment of Autoimmune Diseases.

Tyrosine kinase 2 (TYK2) mediates the interleukin-23 (IL-23), IL-12, and type I interferon (IFN)-driven signal responses that are critical in autoimmune diseases. Here, a series of novel derivatives with an N-(methyl-d 3)pyridazine-3-carboxamide skeleton that bind to the TYK2 pseudokinase domain were designed, synthesized, and evaluated. Among them, compound 30 demonstrated more excellent inhibitory potency against STAT3 phosphorylation than the positive control deucravacitinib. In addition to JAK isoform selectivity, compound 30 exhibited good in vivo and in vitro pharmacokinetic properties. Furthermore, compound 30 was orally highly effective in both IL-23-driven acanthosis and anti-CD40-induced colitis models. Together, these findings support compound 30 as a promising candidate for therapeutic applications in autoimmune diseases.

Magnesium isoglycyrrhizinate prevents cadmium-induced activation of JNK and apoptotic hepatocyte death by reversing ROS-inactivated PP2A.

OBJECTIVES: Cadmium (Cd) induces reactive oxygen species (ROS)-mediated hepatocyte apoptosis and consequential liver disorders. This study aimed to investigate the effect of magnesium isoglycyrrhizinate (MgIG) on Cd-induced hepatotoxicity. METHODS: L02 and AML-12 cells were used to study MgIG hepatoprotective effects. Cd-evoked apoptosis, ROS and protein phosphatase 2A (PP2A)/c-Jun N-terminal kinase (JNK) cascade disruption were analysed by cell viability assay, 6-diamidino-2-phenylindole (DAPI) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, ROS imaging and Western blotting. Pharmacological and genetic approaches were used to explore the mechanisms. KEY FINDINGS: We show that MgIG attenuated Cd-evoked hepatocyte apoptosis by blocking JNK pathway. Pre-treatment with SP600125 or ectopic expression of dominant-negative c-Jun enhanced MgIG's anti-apoptotic effects. Further investigation found that MgIG rescued Cd-inactivated PP2A. Inhibition of PP2A activity by okadaic acid attenuated the MgIG's inhibition of the Cd-stimulated JNK pathway and apoptosis; in contrast, overexpression of PP2A strengthened the MgIG effects. In addition, MgIG blocked Cd-induced ROS generation. Eliminating ROS by N-acetyl-l-cysteine abrogated Cd-induced PP2A-JNK pathway disruption and concurrently reinforced MgIG-conferred protective effects, which could be further slightly strengthened by PP2A overexpression. CONCLUSIONS: Our findings indicate that MgIG is a promising hepatoprotective agent for the prevention of Cd-induced hepatic injury by mitigating ROS-inactivated PP2A, thus preventing JNK activation and hepatocyte apoptosis.

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.

D- and Unnatural Amino Acid Substituted Antimicrobial Peptides With Improved Proteolytic Resistance and Their Proteolytic Degradation Characteristics.

The transition of antimicrobial peptides (AMPs) from the laboratory to market has been severely hindered by their instability toward proteases in biological systems. In the present study, we synthesized derivatives of the cationic AMP Pep05 (KRLFKKLLKYLRKF) by substituting L-amino acid residues with D- and unnatural amino acids, such as D-lysine, D-arginine, L-2,4-diaminobutanoic acid (Dab), L-2,3-diaminopropionic acid (Dap), L-homoarginine, 4-aminobutanoic acid (Aib), and L-thienylalanine, and evaluated their antimicrobial activities, toxicities, and stabilities toward trypsin, plasma proteases, and secreted bacterial proteases. In addition to measuring changes in the concentration of the intact peptides, LC-MS was used to identify the degradation products of the modified AMPs in the presence of trypsin and plasma proteases to determine degradation pathways and examine whether the amino acid substitutions afforded improved proteolytic resistance. The results revealed that both D- and unnatural amino acids enhanced the stabilities of the peptides toward proteases. The derivative DP06, in which all of the L-lysine and L-arginine residues were replaced by D-amino acids, displayed remarkable stability and mild toxicity in vitro but only slight activity and severe toxicity in vivo, indicating a significant difference between the in vivo and in vitro results. Unexpectedly, we found that the incorporation of a single Aib residue at the N-terminus of compound UP09 afforded remarkably enhanced plasma stability and improved activity in vivo. Hence, this derivative may represent a candidate AMP for further optimization, providing a new strategy for the design of novel AMPs with improved bioavailability.