Cyclic Peptide Keap1-Nrf2 Protein-Protein Interaction Inhibitors: Design, Synthesis, and In Vivo Treatment of Acute Lung Injury.

Directly blocking the Keap1-Nrf2 pathway is a promising strategy for the mitigation of acute lung injury (ALI). Peptide Keap1-Nrf2 inhibitors have been reported to have a high Keap1 binding affinity. However, these inhibitors showed weak activity in cells and/or animals. In this study, we designed a series of linear peptides from an Nrf2-based 9-mer Ac-LDEETGEFL-NH2. To improve the cellular activity, we further designed cyclic peptides based on the crystal complex of Keap1 with a linear peptide. Among them, cyclic 9-mer ZC9 targeting Keap1 showed a better affinity (KD2 = 51 nM). Specifically, it exhibited an acceptable water solubility (>38 mg/mL), better cell permeability, cell activity, and metabolic stability (serum t1/2 > 24 h). In the in vitro LPS-induced oxidative damages and ALI model, ZC9 showed significant dose-response reversal activity without apparent toxicity. In conclusion, our results suggested ZC9 as a lead cyclic peptide targeting the Keap1-Nrf2 pathway for ALI clinical treatment.

Total synthesis and structural modification of the dibenzylbutane lignan LCA as a potent anti-inflammatory agent against LPS-induced acute lung injury.

Acute lung injury (ALI) is a serious public health problem associated with high morbidity and mortality. However, few efficacious drugs are clinically available. Inhibition of proinflammatory cytokines is considered to be a promising method for the treatment of inflammatory diseases. Herein, the total synthesis of a dibenzylbutane lignan, 9'-O-di-(E)-feruloyl-meso-5,5'-dimethoxysecoisolariciresinol (LCA), was completed. A series of LCA derivatives were designed and synthesized, and their anti-inflammatory activities were evaluated. Derivative 14r significantly inhibited LPS-induced expression of NO and the proinflammatory cytokines TNF-α, IL-6, and IL-1ß in RAW 264.7 cells and inhibited activation of the NF-κB pathway. Compound 14r reduced LPS-induced pulmonary inflammation and ALI in mice. It showed significant protective effects against LPS-induced ALI in mice and significantly reduced levels of proinflammatory cytokines in serum and bronchoalveolar lavage fluid. The ratio of wet weight to dry weight of lung tissue was normalized by compound 14r, which was consistent with suppression of neutrophil infiltration and production of proinflammatory cytokines. Compound 14r reduced the mRNA expression of some proinflammatory cytokines, improved histopathologic changes, and reduced macrophage infiltration in lung tissues. Collectively, these results suggest a new series of LCA derivatives that could be promising anti-inflammatory agents for ALI treatment.

Further structural optimization and SAR study of sungsanpin derivatives as cell-invasion inhibitors.

Metastasis is one of the major causes of death in patients with cancer, and cell invasion plays a fundamental part in this process. Because of the absence of efficacious treatments, caring for these patients is challenging. Recently, we optimized the structure of the naturally occurring lasso peptide sungsanpin. We identified two peptides, octapeptide S3 and cyclic peptide S4, which inhibited invasion into A549 cells effectively. We undertook an alanine scan of S3 to explore the structure-activity relationship. The linear octapeptide S3-4 and cyclic peptide S4-1 exhibited improved inhibition of invasion into A549 cells. We modified S3-4 to obtain S3-4K, which displayed much higher inhibitory activity against invasion into A549 cells than S3-4. Of all peptides tested, S4-1 upregulated significantly mRNA of tissue inhibitor matrix metalloproteinase TIMP-1 and TIMP-2.

Design, synthesis, evaluation and optimization of novel azole analogues as potent antifungal agents.

In order to develop antifungal drugs, a series of novel azole analogues were designed and synthesized based on our previous work. Most of the target compounds had broad-spectrum antifungal activity, which showed excellent to moderate inhibitory activity against the tested strains, except A. fum 0504656. Among these, compounds B3, B7, B8, B11, B12 and E9 showed excellent activity against C. alb Y0109 and C. alb SC5314 (with the MIC80: 0.0156 ug/mL). In addition, compound B3 showed the best inhibitory activity against fluconazole-resistant strains C. alb 901 and C. alb 904, and had low toxicity against NIH/3T3 cells at the effective MIC range against fungi. Structure-activity relationship and docking studies of the derivatives suggest that the presence of the 2-fluoro-4-hydroxyphenyl and 1,2,3-triazole group enhance the antifungal activity of the compounds, which may be related to the interaction of the key groups with the amino acids surrounding the target enzyme.

SOS1-inspired hydrocarbon-stapled peptide as a pan-Ras inhibitor.

Blocking the interaction between Ras and Son of Sevenless homolog 1 (SOS1) has been an attractive therapeutic strategy for treating cancers involving oncogenic Ras mutations. K-Ras mutation is the most common in Ras-driven cancers, accounting for 86%, with N-Ras mutation and H-Ras mutation accounting for 11% and 3%, respectively. Here, we report the design and synthesis of a series of hydrocarbon-stapled peptides to mimic the alpha-helix of SOS1 as pan-Ras inhibitors. Among these stapled peptides, SSOSH-5 was identified to maintain a well-constrained alpha-helical structure and bind to H-Ras with high affinity. SSOSH-5 was furthermore validated to bind with Ras similarly to the parent linear peptide through structural modeling analysis. This optimized stapled peptide was proven to be capable of effectively inhibiting the proliferation of pan-Ras-mutated cancer cells and inducing apoptosis in a dose-dependent manner by modulating downstream kinase signaling. Of note, SSOSH-5 exhibited a high capability of crossing cell membranes and strong proteolytic resistance. We demonstrated that the peptide stapling strategy is a feasible approach for developing peptide-based pan-Ras inhibitors. Furthermore, we expect that SSOSH-5 can be further characterized and optimized for the treatment of Ras-driven cancers.

Rational Optimizations of the Marine-Derived Peptide Sungsanpin as Novel Inhibitors of Cell Invasion.

Cancer metastasis, including cell invasion, is a major cause of poor clinical outcomes and death in numerous cancer patients. In recent years, many efforts have been made to develop potent therapeutic molecules from naturally derived peptides. Sungsanpin is a naturally derived lasso peptide that inhibits A549 cell invasion. We aimed to evaluate the potential of sungsanpin derivatives as candidates for anti-invasion drugs. We synthesized an analog of sungsanpin (Sun A) using a solid-phase peptide synthesis strategy (SPPS) and further modified its structure to improve its anti-invasion activity. All peptides were tested for their proliferative inhibition and anti-invasion activities in the A549 cell lines. Octapeptide S3 and cyclooctapeptide S4 upregulated the expression of TIMP-1 and TIMP-2 mRNA effectively and thus improved the inhibitory effect on the invasion of A549 cells. The two peptides can inhibit the invasion of A549 cells by up to 60 %, suggesting that they have potential as lead molecules for the development of peptide inhibitors.

Advanced approaches of developing targeted covalent drugs.

In recent years, the development of targeted covalent inhibitors has gained popularity around the world. Specific groups (electrophilic warheads) form irreversible bonds with the side chain of nucleophilic amino acid residues, thus changing the function of biological targets such as proteins. Since the first targeted covalent inhibitor was disclosed in the 1990s, great efforts have been made to develop covalent ligands from known reversible leads or drugs by addition of tolerated electrophilic warheads. However, high reactivity and "off-target" toxicity remain challenging issues. This review covers the concept of targeted covalent inhibition to diseases, discusses traditional and interdisciplinary strategies of cysteine-focused covalent drug discovery, and exhibits newly disclosed electrophilic warheads majorly targeting the cysteine residue. Successful applications to address the challenges of designing effective covalent drugs are also introduced.

Microwave-assisted synthesis of 4-oxo-2-butenoic acids by aldol-condensation of glyoxylic acid.

4-Oxobutenoic acids are useful as biologically active species and as versatile intermediates for further derivatisation. Currently, routes to their synthesis can be problematic and lack generality. Reaction conditions for the synthesis of 4-oxo-2-butenoic acid by microwave-assisted aldol-condensation between methyl ketone derivatives and glyoxylic acid have been developed. They provide the desired products in moderate to excellent yields for a wide range of substrates, by applying a simple procedure to accessible starting materials. The investigation revealed different conditions are required depending on the nature of the methylketone substituent, with aryl derivatives proceeding best using tosic acid and aliphatic substrates reacting best with pyrrolidine and acetic acid. This substituent effect is rationalised by frontier orbital calculations. Overall, this work provides methods for synthesis of 4-oxo-butenoic acids across a broad range of substrates.

FragLites-Minimal, Halogenated Fragments Displaying Pharmacophore Doublets. An Efficient Approach to Druggability Assessment and Hit Generation.

Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource-intensive screening of large libraries of lead-like or fragment molecules. Here, we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.