Design and synthesis of TH19P01-Camptothecin based hybrid peptides inducing effective anticancer responses on sortilin positive cancer cells.

Recently, the sortilin receptor (SORT1) was found to be preferentially over-expressed on the surface of many cancer cells, which makes SORT1 a novel anticancer target. The SORT1 binding proprietary peptide TH19P01 could achieve the SORT1-mediated cancer cell binding and subsequent internalization. Inspired by the peptide-drug conjugate (PDC) strategy, the TH19P01-camptothecin (CPT) conjugates were designed, efficiently synthesized, and evaluated for their anticancer potential in this study. The water solubility, in vitro anticancer activity, time-kill kinetics, cellular uptake, anti-migration activity, and hemolysis effects were systematically estimated. Besides, in order to monitor the release of CPT from conjugates in real-time, the CPT/Dnp-based "turn on" hybrid peptide was designed, which indicted that CPT could be sustainably released from the hybrid peptide in both human serum and cancer cellular environments. Strikingly, compared with free CPT, the water solubility, cellular uptake, and selectivity towards cancer cells of hybrid peptide LYJ-2 have all been significantly enhanced. Moreover, unlike free CPT or TH19P01, LYJ-2 exhibited selective anti-proliferative and anti-migration effects against SORT1-positive MDA-MB-231 cells. Collectively, this study not only established efficient strategies to improve the solubility and anticancer potential of chemotherapeutic agent CPT, but also provided important references for the future development of TH19P01 based PDCs targeting SORT1.

Synthesis and structural optimization of oncolytic peptide LTX-315.

Oncolytic peptides represented potential novel candidates for anticancer treatments especially drug-resistant cancer cell lines. One of the most promising and extensively studied is LTX-315, which is considered as the first in class oncolytic peptide and has entered phase I/II clinical trials. Nevertheless, the shortcomings including poor proteolytic stability, moderate anticancer durability and high synthesis costs may hinder the widespread clinical applications of LTX-315. In order to reduce the synthesis costs, as well as develop derivatives possessing both high protease-stability and durable anticancer efficiency, twenty LTX-315-based derived-peptides were designed and efficiently synthesized. Especially, through solid-phase S-alkylation, as well as the optimized peptide cleavage condition, the derived peptides could be prepared with drastically reduced synthesis cost. The in vitro anticancer efficiency, serum stability, anticancer durability, anti-migration activity, and hemolysis effect were systematically investigated. It was found that derived peptide MS-13 exhibited comparable anticancer efficiency and durability to those of LTX-315. Strikingly, the D-type peptide MS-20, which is the enantiomer of MS-13, was demonstrated to possess significantly high proteolytic stability and sustained anticancer durability. In general, the cost-effective synthesis and stability-guided structural optimizations were conducted on LTX-315, affording the highly hydrolysis resistant MS-20 which possessed durable anticancer activity. Meanwhile, this study also provided a reliable reference for the future optimization of anticancer peptides through the solid-phase S-alkylation and L-type to D-type amino acid substitutions.

d-type peptides based fluorescent probes for "turn on" sensing of heparin.

Developing "turn on" fluorescent probes was desirable for the detection of the effective anticoagulant agent heparin in clinical applications. Through combining the aggregation induced emission (AIE) fluorogen tetraphenylethene (TPE) and heparin specific binding peptide AG73, the promising "turn on" fluorescent probe TPE-1 has been developed. Nevertheless, although TPE-1 could achieve the sensitive and selective detection of heparin, the low proteolytic stability and undesirable poor solubility may limit its widespread applications. In this study, seven TPE-1 derived fluorescent probes were rationally designed, efficiently synthesized and evaluated. The stability and water solubility were systematically estimated. Especially, to achieve real-time monitoring of proteolytic stability, the novel Abz/Dnp-based "turn on" probes that employ the internally quenched fluorescent (IQF) mechanism were designed and synthesized. Moreover, the detection ability of synthetic fluorescent probes for heparin were systematically evaluated. Importantly, the performance of d-type peptide fluorescent probe XH-6 indicated that d-type amino acid substitutions could significantly improve the proteolytic stability without compromising its ability of heparin sensing, and attaching solubilizing tag 2-(2-aminoethoxy) ethoxy) acid (AEEA) could greatly enhance the solubility. Collectively, this study not only established practical strategies to improve both the water solubility and proteolytic stability of "turn on" fluorescent probes for heparin sensing, but also provided valuable references for the subsequent development of enzymatic hydrolysis-resistant d-type peptides based fluorescent probes.

Three Rounds of Stability-Guided Optimization and Systematical Evaluation of Oncolytic Peptide LTX-315.

Oncolytic peptides represent promising novel candidates for anticancer treatments. In our efforts to develop oncolytic peptides possessing both high protease stability and durable anticancer efficiency, three rounds of optimization were conducted on the first-in-class oncolytic peptide LTX-315. The robust synthetic method, in vitro and in vivo anticancer activity, and anticancer mechanism were investigated. The D-type peptides represented by FXY-12 possessed significantly improved proteolytic stability and sustained anticancer efficiency. Strikingly, the novel hybrid peptide FXY-30, containing one FXY-12 and two camptothecin moieties, exhibited the most potent in vitro and in vivo anticancer activities. The mechanism explorations indicated that FXY-30 exhibited rapid membranolytic effects and induced severe DNA double-strand breaks to trigger cell apoptosis. Collectively, this study not only established robust strategies to improve the stability and anticancer potential of oncolytic peptides but also provided valuable references for the future development of D-type peptides-based hybrid anticancer chemotherapeutics.

Design, synthesis and anticancer evaluation of novel oncolytic peptide-chlorambucil conjugates.

Nitrogen mustards (NMs) are an important class of chemotherapeutic drugs and have been widely employed for the treatment of various cancers. However, due to the high reactivity of nitrogen mustard, most NMs react with proteins and phospholipids within the cell membrane. Therefore, only a very small fraction of NMs can reach the reach nucleus, alkylating and cross-linking DNA. To efficiently penetrate the cell membrane barrier, the hybridization of NMs with a membranolytic agent may be an effective strategy. Herein, the chlorambucil (CLB, a kind of NM) hybrids were first designed by conjugation with membranolytic peptide LTX-315. However, although LTX-315 could help large amounts of CLB penetrate the cytomembrane and enter the cytoplasm, CLB still did not readily reach the nucleus. Our previous work demonstrated that the hybrid peptide NTP-385 obtained by covalent conjugation of rhodamine B with LTX-315 could accumulate in the nucleus. Hence, the NTP-385-CLB conjugate, named FXY-3, was then designed and systematically evaluated both in vitro and in vivo. FXY-3 displayed prominent localization in the cancer cell nucleus and induced severe DNA double-strand breaks (DSBs) to trigger cell apoptosis. Especially, compared with CLB and LTX-315, FXY-3 exhibited significantly increased in vitro cytotoxicity against a panel of cancer cell lines. Moreover, FXY-3 showed superior in vivo anticancer efficiency in the mouse cancer model. Collectively, this study established an effective strategy to increase the anticancer activity and the nuclear accumulation of NMs, which will provide a valuable reference for future nucleus-targeting modification of nitrogen mustards.

Efficient synthesis and anticancer evaluation of spider toxin peptide LVTX-8-based analogues with enhanced stability.

Cytotoxic peptides derived from spider venoms have been considered as promising candidates for anticancer treatment. The novel cell penetrating peptide LVTX-8, which is a 25-residue amphipathic α-helical peptide isolated from spider Lycosa vittata, exhibited potent cytotoxicity and is a potential precursor for further anticancer drug development. Nevertheless, LVTX-8 may be easily degraded by multiple proteases, inducing the proteolytic stability problem and short half-life. In this study, ten LVTX-8-based analogs were rationally designed and the efficient manual synthetic method was established by the DIC/Oxyma based condensation system. The cytotoxicity of synthetic peptides was systematically evaluated against seven cancer cell lines. Seven of the derived peptides exhibited high cytotoxicity towards tested cancer in vitro, which was better than or comparable to that of natural LVTX-8. In particular, both N-acetyl and C-hydrazide modified LVTX-8 (825) and the conjugate methotrexate (MTX)-GFLG-LVTX-8 (827) possessed more durable anticancer efficiency, higher proteolytic stability, as well as lower hemolysis. Finally, we confirmed that LVTX-8 could disrupt the integrity of cell membrane, target the mitochondria and reduce the mitochondrial membrane potential to induce the cell death. Taken together, the structural modifications were conducted on LVTX-8 for the first time and the stability significantly improved derivatives 825 and 827 may provide useful references for the modifications of cytotoxic peptides.

Structure-guided peptide engineering of a positive allosteric modulator targeting the outer pore of TRPV1 for long-lasting analgesia.

Transient receptor potential vanilloid 1 (TRPV1) ion channel is a classic analgesic target, but antagonists of TRPV1 failed in clinical trials due to their side effects like hyperthermia. Here we rationally engineer a peptide s-RhTx as a positive allosteric modulator (PAM) of TRPV1. Patch-clamp recordings demonstrate s-RhTx selectively potentiated TRPV1 activation. s-RhTx also slows down capsaicin-induced desensitization of TRPV1 in the presence of calcium to cause more calcium influx in TRPV1-expressing cells. In addition, our thermodynamic mutant cycle analysis shows that E652 in TRPV1 outer pore specifically interacts with R12 and K22 in s-RhTx. Furthermore, we demonstrate in vivo that s-RhTx exhibits long-lasting analgesic effects in noxious heat hyperalgesia and CFA-induced chronic inflammatory pain by promoting the reversible degeneration of intra-epidermal nerve fiber (IENF) expressing TRPV1 channels in mice, while their body temperature remains unaffected. Our results suggest s-RhTx is an analgesic agent as a PAM of TRPV1.

The hybrid oncolytic peptide NTP-385 potently inhibits adherent cancer cells by targeting the nucleus.

The use of oncolytic peptides with activity against a wide range of cancer entities as a new and promising cancer therapeutic strategy has drawn increasing attention. The oncolytic peptide LTX-315 derived from bovine lactoferricin (LfcinB) was found to be highly effective against suspension cancer cells, but not adherent cancer cells. In this study, we tactically fused LTX-315 with rhodamine B through a hybridization strategy to design and synthesize a series of nucleus-targeting hybrid peptides and evaluated their activity against adherent cancer cells. Thus, four hybrid peptides, NTP-212, NTP-217, NTP-223 and NTP-385, were synthesized. These hybrid peptides enhanced the anticancer activity of LTX-315 in a panel of adherent cancer cell lines by 2.4- to 37.5-fold. In model mice bearing B16-F10 melanoma xenografts, injection of NTP-385 (0.5 mg per mouse for 3 consecutive days) induced almost complete regression of melanoma, prolonged the median survival time and increased the overall survival. Notably, the administered dose of NTP-385 was only half the effective dose of LTX-315. We further revealed that unlike LTX-315, which targets the mitochondria, NTP-385 disrupted the nuclear membrane and accumulated in the nucleus, resulting in the transfer of a substantial amount of reactive oxygen species (ROS) from the cytoplasm to the nucleus through the fragmented nuclear membrane. This ultimately led to DNA double-strand break (DSB)-mediated intrinsic apoptosis. In conclusion, this study demonstrates that hybrid peptides obtained from the fusion of LTX-315 and rhodamine B enhance anti-adherent cancer cell activity by targeting the nucleus and triggering DNA DSB-mediated intrinsic apoptosis. This study also provides an advantageous reference for nucleus-targeting peptide modification.

Hybrid peptide NTP-217 triggers ROS-mediated rapid necrosis in liver cancer cells by induction of mitochondrial leakage.

Liver cancer is the third leading cause of cancer-associated mortality globally, and >830,000 patients with liver cancer undergoing treatment succumbed to the disease in 2020, which indicates the urgent need to develop a more effective anti-liver cancer drug. In our previous study, nucleus-targeting hybrid peptides obtained from the fusion of LTX-315 and the rhodamine B group possessed potent anti-adherent cancer cell activity. Hybrid peptides accumulated in the cell nucleus and damaged the nuclear membrane, resulting in the transfer of reactive oxygen species (ROS) from the cytoplasm to the nucleus and the induction of apoptosis. However, the source of the high concentration of ROS within the cytoplasm is unclear. Moreover, although our previous study demonstrated that hybrid peptides possessed potent anticancer activity against adherent cancer cells, their efficacy on liver cancer remained unexplored. The current study found that the hybrid peptide NTP-217 killed liver cancer cells after 4-h treatment with a half-maximal inhibitory concentration of 14.6-45.7 µM. NTP-217 could stably accumulate in the liver tumor tissue and markedly inhibited liver tumor growth in mice. Furthermore, NTP-217 destroyed mitochondria and induced the leakage of mitochondrial contents, resulting in the generation of a substantial quantity of ROS. Unlike the apoptosis induced by 24 h of treatment by NTP-217, 4 h of treatment caused ROS-mediated necrotic cell death. These findings suggested that short-time treatment with hybrid peptides could trigger ROS-mediated rapid necrosis in liver cancer cells, and provided a basis for the future development of hybrid peptides as anti-liver cancer agents.

Comparison of different strategies towards the chemical synthesis of long-chain scorpion toxin AaH-II.

Long-chain scorpion toxin AaH-II isolated from Androctonus australis Hector can selectively inhibit mammalian voltage-gated sodium ion channel Nav 1.7 responsible for pain sensation. Efficient chemical synthesis of AaH-II and its derivatives is beneficial to the study of the function and mechanism of Nav 1.7 and the development of potential peptide inhibitors. Herein, we compared three different strategies, namely, direct solid-phase peptide synthesis, hydrazide-based two-segment native chemical ligation, and hydrazide-based three-segment native chemical ligation for the synthesis of AaH-II. The hydrazide-based two-segment native chemical ligation affords the target toxin with the optimal efficiency, which provides a practically robust procedure for the preparation of tool molecules derived from AaH-II to study the biological functions and modulation of Nav 1.7. Our work highlights the importance of selecting suitable segment condensation approach in the chemical synthesis of protein toxins.

A Diaminodiacid (DADA) Strategy for the Development of Disulfide Surrogate Peptides.

Disulfide bond-containing peptides are useful molecular scaffolds with diagnostic and therapeutic applications due to their good biological activity and good target selectivity, but their utility is sometimes limited by the lability of the disulfide moiety under reducing conditions and in the presence of disulfide bond isomerase. The development of disulfide surrogates with improved redox stability has been an area of ongoing research; and one possible strategy is based on a diaminodiacid (DADA) moiety, which can be used to synthesize the disulfide bond replacement peptides with precise structures and enhanced stability through automated solid-phase peptide synthesis (SPPS). This review summarizes recent developments in the DADA-based SPPS of peptide disulfide surrogates. Some representative applications and structural studies on the DADA-based disulfide surrogates are described.

Hmb(off/on) as a switchable thiol protecting group for native chemical ligation.

A new thiol protecting group Hmb(off/on) is described, which has a switchable activity that may be useful in the chemical synthesis of proteins. When placed on the side chain of Cys, Cys(Hmb(off)) is stable to trifluoroacetic acid (TFA) in the process of solid-phase peptide synthesis. When Cys(Hmb(off)) is treated with neutral aqueous buffers, it is cleanly converted to acid-labile Cys(Hmb(on)), which can later be fully deprotected by TFA to generate free Cys. The utility of Cys(Hmb(off/on)) is demonstrated by the chemical synthesis of an erythropoietin segment, EPO[Cys(98)-Arg(166)]-OH through native chemical ligation.

Blocking of the PD-1/PD-L1 Interaction by a D-Peptide Antagonist for Cancer Immunotherapy.

Blockade of the protein-protein interaction between the transmembrane protein programmed cell death protein 1 (PD-1) and its ligand PD-L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror-image phage display, we developed the first hydrolysis-resistant D-peptide antagonists to target the PD-1/PD-L1 pathway. The optimized compound (D) PPA-1 could bind PD-L1 at an affinity of 0.51 µM in vitro. A blockade assay at the cellular level and tumor-bearing mice experiments indicated that (D) PPA-1 could also effectively disrupt the PD-1/PD-L1 interaction in vivo. Thus D-peptide antagonists may provide novel low-molecular-weight drug candidates for cancer immunotherapy.

Chemical synthesis of proteins using peptide hydrazides as thioester surrogates.

This protocol provides a detailed procedure for the chemical synthesis of proteins through native chemical ligation of peptide hydrazides. The two crucial stages of this protocol are (i) the solid-phase synthesis of peptide hydrazides via Fmoc chemistry and (ii) the native chemical ligation of peptide hydrazides through in situ NaNO2 activation and thiolysis. This protocol may be of help in the synthesis of proteins that are not easily produced by recombinant technology and that include acid-sensitive modifications; it also does not involve the use of hazardous HF. The utility of the protocol is shown for the total synthesis of 140-aa-long α-synuclein, a protein that has an important role in the development of Parkinson's disease. The whole synthesis of the target protein α-synuclein in milligram scale takes ~30 working days.