Discovery of an orally effective double-stapled peptide for reducing ovariectomy-induced bone loss in mice

Stapled peptides with significantly enhanced pharmacological profiles have emerged as promising therapeutic molecules due to their remarkable resistance to proteolysis and performance to penetrate cells.The all-hydrocarbon peptide stapling technique has already widely adopted with great success,yielding numerous potent peptide-based molecules.Based on our prior efforts,we conceived and prepared a double-stapled peptide in this study,termed FRNC-1,which effectively attenuated the bone resorption capacity of mature osteoclasts in vitro through specific inhibition of phosphorylated GSK-3β.The double-stapled peptide FRNC-1 displayed notably improved helical contents and resistance to proteolysis than its linear form.Additionally,FRNC-1 effectively prevented osteoclast activation and improved bone density for ovariectomized(OVX)mice after intravenous injection and importantly,after oral(intragastric)administration.The double-stapled peptide FRNC-1 is the first orally effective peptide that has been validated to date as a therapeutic candidate for postmenopausal osteoporosis(PMOP).

Apoptosis-inducing activity of synthetic hydrocarbon-stapled peptides in H358 cancer cells expressing KRAS^(G12C)

Lung cancers are the leading cause of cancer deaths worldwide and pose a grave threat to human life and health.Non-small cell lung cancer (NSCLC) is the most frequent malignancy occupying80%of all lung cancer subtypes.Except for other mutations (e.g.,KRAS^(G12V/D)) that are also vital for the occurrence,KRAS^(G12C) gene mutation is a significant driving force of NSCLC,with a prevalence of approximately 14% of all NSCLC patients.However,there are only a few therapeutic drugs targeting KRAS^(G12C) mutations currently.Here,we synthesized hydrocarbon-stapled peptide 3 that was much shorter and more stable with modest KRAS^(G12C) binding affinity and the same anti-tumor effect based on the a-helical peptide mimic SAH-SOS1_(A).The stapled peptide 3 effectively induced G2/M arrest and apoptosis,inhibiting cell growth in KRAS-mutated lung cancer cells via disrupting the KRASmediated RAF/MEK/ERK signaling,which was verified from the perspective of genomics and proteomics.Peptide 3 also exhibited strong anti-trypsin and anti-chymotrypsin abilities,as well as good plasma stability and human liver microsomal metabolic stability.Overall,peptide 3 retains the equivalent anti-tumor activity of SAH-SOS1_(A) but with improved stability and affinity,superior to SAH-SOS1_(A).Our work offers a structural optimization approach of KRAS^(G12C) peptide inhibitors for cancer therapy.

A dimethylbromobenzene-cysteine stapled peptide dual inhibitor of the p53-MDM2/MDMX interactions

Aim:Hepatocellular carcinoma(HCC)has emerged as one of the most commonly diagnosed forms of human cancer;yet,the current treatment for HCC is less effective than those used against other cancers.Transcription factor p53 induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress,thereby playing a critical role in protecting cells from malignant transformation.The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53,conferring tumor development and survival.Methods:In this work,we firstly explored the feasibility of antagonists targeting the p53-binding domains of MDM2 and MDMX as a potential method for HCC therapy via the survival rate analysis in The Cancer Genome Atlas.Moreover,we developed a novel stapling strategy for peptide drug design using the reaction between mercapto group and bromine to crosslink the side chains of the two Cys at(i,i+4)positions,and apply it to a series of peptides derived from a dodecameric peptide antagonist of both MDM2 and MDMX,termed p53-MDM2/MDMX inhibitor(PMI).Results:Notably,all of these stapled peptides can compete with p53 for MDM2 or MDMX binding as the similar affinity ;as PMI.More importantly,this stapling functionally rescued PMI that,on its own,failed to activate p53 because of its poor membrane permeability and susceptibility to proteolytic degradation.Conclusion:Taken together,this work not only illustrates that the restoration of p53 is a potentially feasible program for HCC therapy,but promises an important new tool for peptide drug discovery and development for a variety of human diseases.

Structural modification and antitumor activity of antimicrobial peptide HYL

HYL derived from the venom of the solitary bee Hylaeus signatus(Hymenoptera:Colletidae)is anα-helical antimicrobial peptide with 16 residues.To explore whether HYL can be applied in anti-tumor therapy,we synthesized HYL and further modified its structure by using a solid-phase synthesis method,and then evaluated their antitumor activities.Firstly,we identified the key residues of HYL by alanine scanning strategy,and then a series of stapled peptides were synthesized by hydrocarbon stapling strategy without destroying the key residues.All the stapled peptides of HYL showed significant improvement not only inα-helicity,but also in antitumor activity and protease resistance when compared to the parent peptide HYL.The results showed that hydrophobicity and amphiphilicity are important factors affecting the antitumor activity of HYL,and the stapling strategy can significantly affect the proteolytic stability and helicity of HYL.What’s more,we find that the stapled peptides HYL-14,HYL-16 and HYL-18 show a promising prospect for novel anti-tumor drug development.

Stapled SC34EK fusion inhibitors with high potency against HIV-1and improved protease resistance

HIV fusion inhibitors are promising therapeutic agents for AIDS treatment. One fusion inhibitor has been approved as anti-HIV drug, while more of them are in preclinical studies or clinical trials. Highly active fusion inhibitors with excellent pharmacokinetic properties are still needed for development of anti-HIV drugs. We found that all-hydrocarbon staples inserted in SC34 EK could not only enhance the inhibitory activity of inhibitors against HIV-1, but also improve protease resistance. Further study revealed that SC34 EK-1 containing a staple was a potent fusion inhibitor with IC;value of 0.04-6.4 nmol/L towards diverse HIV-1 subtypes and half-life value of 112 min against protease hydrolysis. X-ray crystallography studies indicated that introduction of a hydrocarbon staple in SC34 EK could make the amino acid at the interaction surface form perfect conformation to promote inhibitor peptide interacting with target.

Peptide stapling with the retention of double native side-chains

All-hydrocarbon stapling strategy has been widely applied for enhancing the proteolytic stability of peptides. However, two major technical hurdles to some extent limit the development of stapled peptides for therapeutic usage: rational selection of the stapling sites and the corresponding deletion of the native side chains. Previously we described the development of the olefin-terminated amino acids with the retention of native side chains and successfully applied them in the synthesis of hydrocarbon stapled peptides with single side-chain retention. Here, we explored the feasibility and effectiveness of hydrocarbon stapling strategy characterized as double side-chains retention. Modeled after a lengthy human immunodeficiency virus-1(HIV-1) fusion inhibitor SC34 EK, Leu^(i), Ser^(i+4)and Lys^(i), Leu^(i+4)stapled peptides with the retention of double side-chains were effectively obtained. Our complementary study provided a convenient alternative to address where to install the staple in sequence for conventional all-hydrocarbon peptide stapling. Furthermore, this method not only conferred conformational reinforcement for SC34 EK with high α-helicity and protease resistance, but also preserved the structural characteristic(key peripheral residues, charge and solubility) of the linear peptide to the maximum, which are crucial for anti-HIV-1 activity.

Cysteine-Specific Bioconjugation and Stapling of Unprotected Peptides by Chlorooximes

Cyclic peptides have found applications in fields ranging from drug discovery to nanomaterials.Peptide stapling reagents crosslink two or more residues in peptides to generate macrocycles of diverse topology and introduce linker units that might directly impact the properties and biological functions of cyclic peptides.Herein,we demonstrate that chlorooxime derivatives are cysteine-specific peptide bioconjugation and stapling reagents that generate stable thiohydroximate linkages.