Advances in biosynthesis of peptide drugs: Technology and industrialization.

Peptide drugs are developed from endogenous or synthetic peptides with specific biological activities. They have advantages of strong target specificity, high efficacy and low toxicity, thus showing great promise in the treatment of many diseases such as cancer, infections, and diabetes. Although an increasing number of peptide drugs have entered market in recent years, the preparation of peptide drug substances is yet a bottleneck problem for their industrial production. Comparing to the chemical synthesis method, peptide biosynthesis has advantages of simple synthesis, low cost, and low contamination. Therefore, the biosynthesis technology of peptide drugs has been widely used for manufacturing. Herein, we reviewed the development of peptide drugs and recent advances in peptide biosynthesis technology, in order to shed a light to the prospect of industrial production of peptide drugs based on biosynthesis technology.

Self-Assembly Nanostructure of Myristoylated ω-Conotoxin MVIIA Increases the Duration of Efficacy and Reduces Side Effects.

Chronic pain is one of the most prevalent health problems worldwide. An alternative to suppress or alleviate chronic pain is the use of peptide drugs that block N-type Ca2+ channels (Cav2.2), such as ω-conotoxin MVIIA. Nevertheless, the narrow therapeutic window, severe neurological side effects and low stability associated with peptide MVIIA have restricted its widespread use. Fortunately, self-assembly endows the peptide with high stability and multiple functions, which can effectively control its release to prolong its duration of action. Inspired by this, MVIIA was modified with appropriate fatty acid chains to render it amphiphilic and easier to self-assemble. In this paper, an N-terminal myristoylated MVIIA (Myr-MVIIA, medium carbon chain length) was designed and prepared to undergo self-assembly. The present results indicated that Myr-MVIIA can self-assemble into micelles. Self-assembled micelles formed by Myr-MVIIA at higher concentrations than MVIIA can prolong the duration of the analgesic effect and significantly reduce or even eliminate the side effects of tremor and coordinated motor dysfunction in mice.

A single-chain variable fragment-anticancer lytic peptide (scFv-ACLP) fusion protein for targeted cancer treatment.

Antibody-directed drugs for targeted cancer treatment have become a hot topic in new anticancer drug development; however, antibody-fused therapeutic peptides were rarely documented. Herein, we designed a fusion protein with a cetuximab-derived single-chain variable fragment targeting epidermal growth factor receptor (anti-EGFR scFv) and the anticancer lytic peptide (ACLP) ZXR2, connected by a linker (G4 S)3 and MMP2 cleavage site. The anti-EGFR scFv-ZXR2 recombinant protein showed specific anticancer activity on EGFR-overexpressed cancer cell lines in a concentration- and time-dependent manner, as it can bind to EGFR on cancer cell surfaces. This fusion protein caused cell membrane lysis as ZXR2, and showed improved stability in serum compared with ZXR2. These results suggest that scFv-ACLP fusion proteins may be potential anticancer drug candidates for targeted cancer treatment, which also provide a feasible idea for targeted drug design.

Computer-Aided Design of Lasso-like Self-Assembling Anticancer Peptides with Multiple Functions for Targeted Self-Delivery and Cancer Treatments.

Anticancer peptides are promising drug candidates for cancer treatment, but the short circulation time and low delivery efficiency limit their clinical applications. Herein, we designed several lasso-like self-assembling anticancer peptides (LASAPs) integrated with multiple functions by a computer-aided approach. Among these LASAPs, LASAP1 (CRGDKGPDCGKAFRRFLGALFKALSHLL, 1-9 disulfide bond) was determined to be superior to the others because it can self-assemble into homogeneous nanoparticles and exhibits improved stability in serum. Thus, LASAP1 was chosen for proving the design idea. LASAP1 can self-assemble into nanoparticles displaying iRGD on the surface because of its amphiphilic structure and accumulate to the tumor site after injection because of the EPR effect and iRGD targeting to αVß3 integrin. The nanoparticles could disassemble in the acidic microenvironment of the solid tumor, and cleaved by the overexpressed hK2, which was secreted by prostate tumor cells, to release the effector peptide PTP-7b (FLGALFKALSHLL), which was further activated by the acidic pH. Therefore, LASAP1 could target the orthotopic prostate tumor in the model mice after intraperitoneal injection and specifically inhibit tumor growth, with low systematic toxicity. Combining the multiple targeting functions, LASAP1 represents a promising design of self-delivery of peptide drugs for targeted cancer treatments.

Phase separation of p53 induced by its unstructured basic region and prevented by oncogenic mutations in tetramerization domain.

Liquid-liquid phase separation (LLPS) drives the formation of extensive membrane-less compartments to regulate various cellular biological activities both physiologically and pathologically. It has been widely accepted that LLPS is closely related to amyloid diseases and increasing reports have linked this phenomenon to cancers. Mutations of tumor suppressor protein p53 exist in more than half of malignant tumors, making the protein vitally important in cancer research. Recently, p53 was reported to undergo phase separation, which may regulate the function of p53. The molecular mechanism of p53 phase separation and how this process relates to cancer remains largely unclear. Herein, we find that the disordered unstructured basic region (UBR) plays a crucial role in p53 LLPS, driven by electrostatic and hydrophobic interactions. Mutations in the tetramerization domain (TD) disrupt p53 phase separation by preventing the tetramer formation. Furthermore, our results have revealed that, in response to DNA damage in cell, the wild type (WT) p53 undergoes LLPS, while LLPS in oncogenic mutations is diminished or eliminated. The expression of the target gene of p53 decreased significantly with the mutations and cell survival increased with the mutations. Thus, we propose a novel mechanism of p53 carcinogenesis, whereby oncogenic mutations in TD impair the formation of p53 condensates, decreasing the activation of target genes and promoting cancer progression. This study helps to understand the behavior and function of p53 in a different aspect and may provide insights into cancer therapies targeting p53.

Self-assembling branched amphiphilic peptides for targeted delivery of small molecule anticancer drugs.

Water insolubility poses a significant challenge in the clinical applications of many small molecule drugs. To improve the drug delivery efficiency, two branched amphiphilic peptides (BAPs) were designed in a computer-aided manner, for drug-loading through peptide self-assembling. The structures of the two BAPs, bis(LVFFA)-K-RGD (PepV-1) and bis(FHF)-K-RGD (PepV-2), were inspired by phospholipids, containing the RGD sequence as the hydrophilic head and two hydrophobic sequences as the hydrophobic tails. PepV-1 could self-assemble into nano-fibrils with a hydrophobic core and the RGD moiety on the surface. Its drug-loading efficiency (DE%) of three small molecule anticancer drugs (doxorubicin, camptothecin and curcumin) ranged from 9.90% to 11.74%, and entrapment efficiency (EE%) ranged from 37.30% to 43.00%. Pep-V2 could self-assemble into bilayer delimited nano-vesicles. The DE% of PepV-2 for these drugs ranged from 15.87% to 18.55%, and the EE% ranged from 60.45% to 73.23%. Both BAP carriers could prolong the release of the small molecule drugs, and the PepV-2 vesicles also showed pH-triggered increase of drug release due to the histidine residues. Bothe BAP carriers could increase the cytotoxicity against cancer cells, which might be due to the targeting on the cancer overexpressed integrins. The designed BAP carriers represent promising functional drug carriers for targeted drug delivery, and will be useful for improving the clinical use of small molecule drugs, especially for those with poor water solubility.

Novel branched amphiphilic peptides for nucleic acids delivery.

Highly efficient and safe non-viral vectors for nucleic acids delivery have attracted much attention due to their potential applications in gene therapy, gene editing and vaccination against infectious diseases, and various materials have been investigated and designed as delivery vectors. Herein, we designed a series of branched amphiphilic peptides (BAPs) and tested their applications as pDNA/mRNA delivery vectors. The BAP structure was inspired by the phospholipids, in which lysine oligomers were used as the "polar head", segments containing phenylalanine, histidine and leucine were used as the "hydrophobic tails", and a lysine residue was used as the branching point. By comparing the gel retardation, particle sizes and zeta potentials of the BAP/pDNA complexes of the short-branch BAPs (BAP-V1 âˆ¼ BAP-V4), we determined the optimal lysine oligomer was K6. However, their cell transfection efficiencies were not satisfactory, and thus three long-branch BAPs (BAP-V5 âˆ¼ BAP-V7) were further designed. In these long-branch BAPs, more hydrophobic residues were added and the overall amphiphilicity increased accordingly. The results showed that these three BAPs could effectively compact the nucleic acids, including both pDNA and mRNA, and all could transfect nucleic acids into HEK 293 cells, with low cytotoxicity. Among the three long-branch BAPs, BAP-V7 (bis(FFLFFHHH)-K-K6) showed the best transfection efficiency at N/P = 10, which was better than the commercial transfection reagent PEI-25 K. These results indicate that increased amphiphilicity would also benefit for BAP mediated nucleic acid delivery. The designed BAPs provide more documents of such novel type of nucleic acids delivery vectors, which is worth of further investigation as a new gene theranostic platforms.

Intrinsically Disordered Protein Condensate-Modified Surface for Mitigation of Biofouling and Foreign Body Response.

Mitigation of biofouling and the host's foreign body response (FBR) is a critical challenge with biomedical implants. The surface coating with various anti-fouling materials provides a solution to overcome it, but limited options in clinic and their potential immunogenicity drive the development of more alternative coating materials. Herein, inspired by liquid-liquid phase separation of intrinsically disordered proteins (IDPs) to form separated condensates in physiological conditions, we develop a new type of low-fouling biomaterial based on flexible IDP of FUS protein containing rich hydrophilic residues. A chemical structure-defined FUS IDP sequence tagged with a tetra-cysteine motif (IDPFUS) was engineered and applied for covalent immobilization on various surfaces to form a uniform layer of protein tangles, which boosted strong hydration on surfaces, as revealed by molecular dynamics simulation. The IDPFUS-coated surfaces displayed excellent performance in resisting adsorption of various proteins and adhesion of different cells, platelets, and bacteria. Moreover, the IDPFUS-coated implants largely mitigated the host's FBR compared with bare implants and particularly outperformed PEG-coated implants in reducing collagen encapsulation. Thus, this novel low-fouling and anti-FBR strategy provides a potential surface coating material for biomedical implants, which will also shed light on exploring similar applications of other IDP proteins.

Virtual Evolution of HVEM Segment for Checkpoint Inhibitor Discovery.

Immune therapy has emerged as an effective treatment against cancers. Inspired by the PD-1/PD-L1 antibodies, which have achieved great success in clinical, other immune checkpoint proteins have drawn increasing attention in cancer research. B and T lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) are potential targets for drug development. The co-crystal structure of BTLA/HVEM have revealed that HVEM (26-38) fragment is the core sequence which directly involved on the interface. Herein, we conducted virtual evolution with this sequence by using saturation mutagenesis in silico and mutants with lower binding energy were selected. Wet-lab experiments confirmed that several of them possessed higher affinity with BTLA. Based on the best mutant of the core sequence, extended peptides with better efficacy were obtained. Furthermore, the mechanism of the effects of mutations was revealed by computational analysis. The mutated peptide discovered here can be a potent inhibitor to block BTLA/HVEM interaction and its mechanism may extend people's view on inhibitor discovery for the checkpoint pair.

Computational design of ultrashort peptide inhibitors of the receptor-binding domain of the SARS-CoV-2 S protein.

Targeting the interaction between severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) is believed to be an effective strategy for drug design to inhibit the infection of SARS-CoV-2. Herein, several ultrashort peptidase inhibitors against the RBD-ACE2 interaction were obtained by a computer-aided approach based on the RBD-binding residues on the protease domain (PD) of ACE2. The designed peptides were tested on a model coronavirus GX_P2V, which has 92.2 and 86% amino acid identity to the SARS-CoV-2 spike protein and RBD, respectively. Molecular dynamics simulations and binding free energy analysis predicted a potential binding pocket on the RBD of the spike protein, and this was confirmed by the specifically designed peptides SI5α and SI5α-b. They have only seven residues, showing potent antiviral activity and low cytotoxicity. Enzyme-linked immunosorbent assay result also confirmed their inhibitory ability against the RBD-ACE2 interaction. The ultrashort peptides are promising precursor molecules for the drug development of Corona Virus Disease 2019, and the novel binding pocket on the RBD may be helpful for the design of RBD inhibitors or antibodies against SARS-CoV-2.

Amyloid-Forming Segment Induces Aggregation of FUS-LC Domain from Phase Separation Modulated by Site-Specific Phosphorylation.

The RNA-binding protein fused in sarcoma (FUS) forms physiological granules and pathological fibrils, which facilitate RNA functions and cause neurodegenerative diseases, respectively. Phosphorylation at Ser/Thr residues may regulate the functional assembly of FUS and prevent pathological aggregation in cells. However, the low-complexity nature of the FUS sequence makes it challenging to characterize how phosphorylation of specific sites within the core amyloid-forming segment affects aggregation. Taking advantage of the recently solved molecular structures of the fibrillar core of the FUS low-complexity (FUS-LC) domain, we systematically investigated the aggregation of repeated segments within the core. We identified a segment with a strong amyloid-forming tendency that induced the aggregation of FUS-LC domain in phase-separated liquid droplets and further seeded the aggregation of full-length FUS. The aggregation propensity and seeding ability of this amyloid-forming segment were modulated by site-specific phosphorylation. Solid-state nuclear magnetic resonance (NMR) spectroscopy and computational modeling implied that site-specific phosphorylation at Ser61 plays key roles in FUS assembly by disrupting both intra- and intermolecular interactions that maintain the amyloid core structure.

Fused in Sarcoma: Properties, Self-Assembly and Correlation with Neurodegenerative Diseases.

Fused in sarcoma (FUS) is a DNA/RNA binding protein that is involved in RNA metabolism and DNA repair. Numerous reports have demonstrated by pathological and genetic analysis that FUS is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and polyglutamine diseases. Traditionally, the fibrillar aggregation of FUS was considered to be the cause of those diseases, especially via its prion-like domains (PrLDs), which are rich in glutamine and asparagine residues. Lately, a nonfibrillar self-assembling phenomenon, liquid-liquid phase separation (LLPS), was observed in FUS, and studies of its functions, mechanism, and mutual transformation with pathogenic amyloid have been emerging. This review summarizes recent studies on FUS self-assembling, including both aggregation and LLPS as well as their relationship with the pathology of ALS, FTLD, and other neurodegenerative diseases.

A comparison of mesoporous silica nanoparticles and mesoporous organosilica nanoparticles as drug vehicles for cancer therapy.

Mesoporous silica nanoparticles (MSNs) are promising drug carriers for use in cancer treatment owing to their excellent biocompatibility and drug-loading capacity. However, MSN's incomplete drug release and toxic bioaccumulation phenomena limit their clinical application. Recently, researchers have presented redox responsive mesoporous organosilica nanoparticles containing disulfide (S-S) bridges (ss-MONs). These nanoparticles retained their ability to undergo structural degradation and increased their local release activity when exposed to reducing agents. Disulfide-based mesoporous organosilica nanoparticles offer researchers a better option for loading chemotherapeutic drugs due to their effective biodegradability through the reduction of glutathione. Although the potential of ss-MONs in cancer theranostics has been studied, few researchers have systematically compared ss-MONs with MSNs with regard to endocytosis, drug release, cytotoxicity, and therapeutic effect. In this work, ss-MONs and MSNs with equal morphology and size were designed and used to payload doxorubicin hydrochloride (DOX) for liver cancer chemotherapy. The ss-MONs showed considerable degradability in the presence of glutathione and performed comparably to MSNs on biocompatibility measures, including cytotoxicity and endocytosis, as well as in drug-loading capacity. Notably, DOX-loaded ss-MONs exhibited higher intracellular drug release in cancer cells and better anticancer effects in comparison with DOX-loaded MSNs. Hence, the ss-MONs may be more desirable carriers for a highly efficient and safe treatment of cancer.

Cancer cell membrane-modified biodegradable mesoporous silica nanocarriers for berberine therapy of liver cancer.

Berberine (Ber) is regarded as a new, active and natural anti-cancer product; however, its clinical application has been limited due to its low aqueous solubility, poor gastrointestinal absorption, short residence time and poor targeting abilities. Hence, we reported a biomimetic nanoparticle as a drug delivery system to surmount these obstacles. We fabricated disulfide (S-S)-bridged mesoporous organosilica nanoparticles (ss-MONs) for Ber loading, which possessed uniform morphology, controllable mesoporous properties, highly-efficient drug loading capacity and superior biocompatibility. More interestingly, ss-MONs exhibited effective biodegradability under glutathione conditions through the breakage of the disulfide bond in ss-MONs, which promoted the Ber release. After coating human liver cancer HepG2 cell membranes (CM) on the surface of ss-MONs, the obtained CM-ss-MONs-Ber enhanced accumulation in liver cancer tissue through homologous targeting and effectively avoiding rapid blood clearance. Our findings indicate that CM-ss-MONs might be desirable drug carriers to promote the clinical use of Ber against liver cancer.

High-Resolution Insights into the Stepwise Self-Assembly of Nanofiber from Bioactive Peptides.

Peptide self-assembly has a profound biological significance since self-assembled bioactive peptides are gifted with improved bioactivity as well as life-span. In this study, peptide self-assembly was investigated using a therapeutic peptide, PTP-7S (EENFLGALFKALSKLL). Combining experiments of atomic force microscopy (AFM), circular dichroism (CD), and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence spectra, PTP-7S showed the α-helical structure and was found self-assembling into nanofibers in solution. Relying on the coarse-grained (CG) dynamic simulations, the self-assembling of PTP-7S was revealed as a stepwise process that peptide monomers first clustered into peptide-assembling units (PUs) with charged surface, and then the PUs integrated together to construct nanofibril aggregates. Different roles of the nonbonded driving forces did play in the two phases: the hydrophobic force and electrostatic interaction acted as the predominant motivations in the formation of PUs and nanofiber, respectively. Moreover, the electrostatic interaction helped to guide the longitudinal growth of peptide nanofibers. A sequence principle is proposed for peptide self-assembling in aqueous solution: a balance of the counter charges and sufficient hydrophobicity degree. The self-assembled PTP-7S displayed good anticancer activity, proteases resistance, and sustained drug-release, showing a great potential for clinical application. This study reveals the molecular mechanism in explaining PTP-7S self-assembly and it is beneficial for future innovation of the self-assembled bioactive peptides.

Self-assembly nanostructure controlled sustained release, activity and stability of peptide drugs.

Peptides are considered as a new generation of drugs due to their high structural and functional diversity. However, the development of peptide drugs is always limited by their poor stability and short circulation time. Carriers are applied for peptide drug delivery, but there may be problems like poor loading efficiency and undesired xenobiotic toxicity. Peptide self-assembly is an effective approach to improve the stability and control the release of peptide drugs. In this study, two self-assembling anticancer peptides are designed by appending a pair of glutamic acid and asparagine to either the N-terminus or the C-terminus of a lytic peptide. This simple, yet rational sequence modification was made to change the amphiphilic pattern and secondary structural content of the parent peptide, thereby modulating its self-assembly process. It was found that the N-terminus modified peptide favors the formation of nanofibrils and the peptide with C-terminal modification formed micelles. Although both nanostructures showed prolonged action profiles and improved serum stability compared to the parent peptide, the morphology of the nanostructures is highly critical to manipulate the release profile of the free peptide from the assembly and regulate their bioactivity. We believe the self-assembly approach demonstrated in this study can be applied to a variety of therapeutic peptide drugs to improve their stability and therapeutic activity for the development of carrier-free drug delivery system.

Isoleucine/leucine residues at "a" and "d" positions of a heptad repeat sequence are crucial for the cytolytic activity of a short anticancer lytic peptide.

Many lytic peptides contain a heptad sequence with leucine or isoleucine residues at "a" and "d" positions. However, their roles in the peptide-induced cytolytic process remain unclear. We have recently reported an anticancer lytic peptide ZXR-2 (FKIGGFIKKLWRSLLA), which contains a shortened zipper-like sequence with Ile/Leu at "a" and "d" positions. To understand the roles of these Ile/Leu residues, a series of analogs were constructed by sequentially replacing the Ile or Leu residue with alanine (Ala). Significant reduction of the cytolytic activity was observed when the Ile (3rd and 7th) and Leu (10th and 14th) residues at the "a" and "d" positions were substituted, while the replacement of the separate Leu (15th) residue had less effect. Based on the quenching of the intrinsic fluorescence of the peptides and their induced surface pressure changes of lipid monolayer, it was conjectured that the peptide ZXR-2 might insert into cell membranes from the C-terminal and to a depth of the W11 position. Accordingly, I3, I7, and L10 residues which mainly exposed in aqueous solution were more responsible for the peptide self-association on cell membranes, while L14, together with L15, might help peptide insert and anchor to cell membranes. These results are significant to elucidate the crucial roles of such Ile/Leu residues at "a" and "d" positions in peptide-peptide and peptide-membrane interactions to exert the membrane disruption activity of lytic peptides. With further understanding about the structure-activity relationship of lytic peptides, it would be helpful for designing novel anticancer lytic peptides.

How charge distribution influences the function of membrane-active peptides: Lytic or cell-penetrating?

Lytic and cell-penetrating peptides (CPPs) are both membrane-active peptides sharing similar physicochemical properties. Although their respective functions have been intensively investigated, the difference of intrinsic properties between these two types of peptides is rarely discussed. In this study, we designed a series of analogs of a recently discovered CPP ZXR-1 (FKIGGFIKKLWRSKLA) by varying the charge distributions both on the helical wheel projection and along the sequence. These peptides showed different functions on cell membranes, including membrane lytic (peptide Z1), cell-penetrating (peptide ZXR-1, Z2 and Z3), and inactive (peptide Z4) peptides. The three groups of peptides displayed different interactions with model lipid monolayer, and found that peptide insertion might be an important dynamic step to distinguish lytic and cell penetrating functions. Based on the analysis of charge distribution patterns, it was proposed that the charge distributions on the helical wheel and along the sequence are both able to influence the functions of the membrane-active peptides. This finding provides a further understanding about the effect of charge distribution on the functions of membrane-active peptides, and will be helpful for the design of functional peptides.

From a pro-apoptotic peptide to a lytic peptide: One single residue mutation.

Further discovery and design of new anticancer peptides are important for the development of anticancer therapeutics, and study on the detailed acting mechanism and structure-function relationship of peptides is critical for anticancer peptide design and application. In this study, a novel anticancer peptide ZXR-1 (FKIGGFIKKLWRSKLA) derived from a known anticancer peptide mauriporin was developed, and a mutant ZXR-2 (FKIGGFIKKLWRSLLA) with only one residue difference at the 14th position (Lys→Leu) was also engineered. Replacement of the lysine with leucine made ZXR-2 more potent than ZXR-1 in general. Even with only one residue mutation, the two peptides displayed distinct anticancer modes of action. ZXR-1 could translocate into cells, target on the mitochondria and induce cell apoptosis, while ZXR-2 directly targeted on the cell membranes and caused membrane lysis. The variance in their acting mechanisms might be due to the different amphipathicity and positive charge distribution. In addition, the two Ile-Leu pairs (3-10 and 7-14) in ZXR-2 might also play a role in improving its cytotoxicity. Further study on the structure-function relationship of the two peptides may be beneficial for the design of novel anticancer peptides and peptide based therapeutics.

An aptamer based surface plasmon resonance biosensor for the detection of ochratoxin A in wine and peanut oil.

Ochratoxin A (OTA), as a kind of chlorophenolic mycotoxin, exist widely in plant origin food and is harmful to human. Herein, a surface plasmon resonance (SPR) biosensor using an anti-OTA aptamer immobilized sensor chip was developed to measure ochratoxin A (OTA) quantificationally through a straightforward direct binding assay. The streptavidin protein as a crosslinker was immobilized onto the surface of a sensor chip and the biotin-aptamer was captured through streptavidin-biotin interaction. The biosensor exhibited a detection range from 0.094 to 100ng/mL (linear range from 0.094 to 10ng/mL) of OTA with a lower detection limit of 0.005ng/mL. Detection of OTA in wine and peanut oil was further performed in the SPR biosensor using simple liquid-liquid extraction for sample pretreatments. Recoveries of ochratoxin A from spiked samples ranged from 86.9% to 116.5% and coefficients of variation (CVs) ranged from 0.2% to 6.9%. The developed methods in our studies showed good analytical performances with limits of detection much lower than the maximum residue limit, as well as a good reproducibility and stability.