High-affinity Peptide Research Articles

Discovery of TIGIT inhibitors by phage display

Abstract TIGIT is an emerging immune checkpoint receptor expressed higher than PD-1 in some tumors especially anti-PD1 resistant tumors. By competing with the co-stimulatory receptor CD226/DNAM-1 for binding to CD155/PVR, TIGIT transduces inhibitory signals and suppresses the immune response. Therefore, we sought to discover TIGIT inhibitors for improving tumor immunotherapy. Through directional cloning, bacterial transformation and phage amplification techniques, we generated a novel genetically encoded, phage-displayed bicyclic peptide library comprised of more than a million peptide sequences. Depending on the two combinations of different disulfide bonds, each peptide backbone is predicted to be folded into either endo-bicyclic or exo-bicyclic structure. We conducted three rounds of phage display panning against recombinant TIGIT IgV domain that enriched high-affinity peptides and identified promising TIGIT binding peptides. Phage ELISA showed binding of phage-presented peptides to recombinant TIGIT IgV and competitive ELISA evaluated the ability of the peptides to disrupt interactions between TIGIT and PVR. This work validated the efficiency of the phage-displayed bicyclic peptide library for discovery of ligands targeting protein receptors and led to identification of novel TIGIT inhibitors. AAI Careers in Immunology Fellowship

Rapid Evaluation of Staple Placement in Stabilized α Helices Using Bacterial Surface Display.

There are a wealth of proteins involved in disease that cannot be targeted by current therapeutics because they are inside cells, inaccessible to most macromolecules, and lack small-molecule binding pockets. Stapled peptides, where two amino acids are covalently linked, form a class of macrocycles that have the potential to penetrate cell membranes and disrupt intracellular protein-protein interactions. However, their discovery relies on solid-phase synthesis, greatly limiting queries into their complex design space involving amino acid sequence, staple location, and staple chemistry. Here, we use stabilized peptide engineering by Escherichia coli display (SPEED), which utilizes noncanonical amino acids and click chemistry for stabilization, to rapidly screen staple location and linker structure to accelerate peptide design. After using SPEED to confirm hotspots in the mdm2-p53 interaction, we evaluated different staple locations and staple chemistry to identify several novel nanomolar and sub-nanomolar antagonists. Next, we evaluated SPEED in the B cell lymphoma 2 (Bcl-2) protein family, which is responsible for regulating apoptosis. We report that novel staple locations modified in the context of BIM, a high affinity but nonspecific naturally occurring peptide, improve its specificity against the highly homologous proteins in the Bcl-2 family. These compounds demonstrate the importance of screening linker location and chemistry in identifying high affinity and specific peptide antagonists. Therefore, SPEED can be used as a versatile platform to evaluate multiple design criteria for stabilized peptide engineering.

Harnessing protein sensing ability of electrochemical biosensors via a controlled peptide receptor–electrode interface

BackgroundCathepsin B, a cysteine protease, is considered a potential biomarker for early diagnosis of cancer and inflammatory bowel diseases. Therefore, more feasible and effective diagnostic method may be beneficial for monitoring of cancer or related diseases.ResultsA phage-display library was biopanned against biotinylated cathepsin B to identify a high-affinity peptide with the sequence WDMWPSMDWKAE. The identified peptide-displaying phage clones and phage-free synthetic peptides were characterized using enzyme-linked immunosorbent assays (ELISAs) and electrochemical analyses (impedance spectroscopy, cyclic voltammetry, and square wave voltammetry). Feasibilities of phage-on-a-sensor, peptide-on-a-sensor, and peptide-on-a-AuNPs/MXene sensor were evaluated. The limit of detection and binding affinity values of the peptide-on-a-AuNPs/MXene sensor interface were two to four times lower than those of the two other sensors, indicating that the peptide-on-a-AuNPs/MXene sensor is more specific for cathepsin B (good recovery (86–102%) and %RSD (< 11%) with clinical samples, and can distinguish different stages of Crohn’s disease. Furthermore, the concentration of cathepsin B measured by our sensor showed a good correlation with those estimated by the commercially available ELISA kit.ConclusionIn summary, screening and rational design of high-affinity peptides specific to cathepsin B for developing peptide-based electrochemical biosensors is reported for the first time. This study could promote the development of alternative antibody-free detection methods for clinical assays to test inflammatory bowel disease and other diseases.

Machine Learning Guided Design of High-Affinity ACE2 Decoys for SARS-CoV-2 Neutralization.

A potential therapeutic strategy for neutralizing SARS-CoV-2 infection is engineering high-affinity soluble ACE2 decoy proteins to compete for binding to the viral spike (S) protein. Previously, a deep mutational scan of ACE2 was performed and has led to the identification of a triple mutant variant, named sACE22.v.2.4, that exhibits subnanomolar affinity to the receptor-binding domain (RBD) of S. Using a recently developed transfer learning algorithm, TLmutation, we sought to identify other ACE2 variants that may exhibit similar binding affinity with decreased mutational load. Upon training a TLmutation model on the effects of single mutations, we identified multiple ACE2 double mutants that bind SARS-CoV-2 S with tighter affinity as compared to the wild type, most notably L79V;N90D that binds RBD similarly to ACE22.v.2.4. The experimental validation of the double mutants successfully demonstrates the use of machine learning approaches for engineering protein-protein interactions and identifying high-affinity ACE2 peptides for targeting SARS-CoV-2.

What does alphafold know about protein folding and peptide binding.

Machine learning approaches have recently revolutionized structural biology by predicting 3D structures of proteins at atomistic level from their sequence with high confidence and accuracy. The community is pushing the limits of interpretability and application of these algorithms beyond their original objective. Here, we present our studies on what deep neural network models for protein structure prediction such as AlphaFold can shed light on two fundamental biological processes: (1) protein folding and (2) protein-peptide binding. We first demonstrate our studies on deciphering the folding mechanism of protein G, L and their mutants, two pair of proteins with low sequence similarity that fold into the same topology. By using large-scale molecular dynamics simulations with advanced enhanced sampling methods and Markov state modeling analysis, we observed how the sequence difference impacts folding pathways and rate in unprecedented detail. Then, we show a novel fragment decomposition approach using AlphaFold to identify preferences for secondary structure element combinations that follow the order of events observed in the folding pathways. Standing upon the highly sensitive mapping of AlphaFold between the sequence and structure, we discovered how to directly use the model to identify high affinity peptide binders using a competitive binding assay. For systems in which the individual structures of the peptides are well predicted, the assay captures the higher affinity binder in the bound state, and the other peptide in the unbound form. The speed and robustness of the method will make it readily applicable in screening libraries of peptide sequences to prioritize for detailed experimental characterization.

Virtual Screening-Based Peptides Targeting Spike Protein to Inhibit Porcine Epidemic Diarrhea Virus (PEDV) Infection.

Due to the rapid mutation of porcine epidemic diarrhea virus (PEDV), existing vaccines cannot provide sufficient immune protection for pigs. Therefore, it is urgent to design the affinity peptides for the prevention and control of this disease. In this study, we made use of a molecular docking technology for virtual screening of affinity peptides that specifically recognized the PEDV S1 C-terminal domain (CTD) protein for the first time. Experimentally, the affinity, cross-reactivity and sensitivity of the peptides were identified by an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR) test, separately. Subsequently, Cell Counting Kit-8 (CCK-8), quantitative real-time PCR (qRT-PCR), Western blot and indirect immunofluorescence were used to further study the antiviral effect of different concentrations of peptide 110766 in PEDV. Our results showed that the P/N value of peptide 110766 at 450 nm reached 167, with a KD value of 216 nM. The cytotoxic test indicated that peptide 110766 was not toxic to vero cells. Results of the absolute quantitative PCR revealed that different concentrations (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) of peptide 110766 could significantly reduce the viral load of PEDV compared with the virus group (p < 0.0001). Similarly, results of Western blot and indirect immunofluorescence also suggested that the antiviral effect of peptide 110766 at 3.125 is still significant. Based on the above research, high-affinity peptide 110766 binding to the PEDV S1-CTD protein was attained by a molecular docking technology. Therefore, designing, screening, and identifying affinity peptides can provide a new method for the development of antiviral drugs for PEDV.

Discovery, development and implementation of biomarker-specific peptide BRE for biosensing hydrocarbon-degrading fungi

Fuel biocontamination is a significant problem that demands new tools for detection and mitigation. Due to the complexity of fuel microbiome, it is critical to differentiate the type of microorganisms present to apply appropriate countermeasures, and fuel-degrading fungi are among the main culprits of biodeterioration. To establish robust biosensors for detection of fuel biocontaminants, a conserved cell surface biomarker must be identified before selection of high-affinity peptide biorecognition elements (BRE) can be screened. To address this need, we applied functional genomics and transcriptomics to characterize a conserved cell surface protein of fuel-degrading fungi, Cytochrome P450 Alkane Hydroxylase (Alk), which is highly expressed during growth in fuel and is critical for alkane degradation. Multiple conserved extracellular ALK epitopes were used to screen a phage-displayed peptide library and a single BRE (5ALK-23) was identified. Molecular and fluorescence assays demonstrated high affinity and specificity of 5ALK-23 for fungi and a low limit of detection that ranged from 1 × 101 cells/mL to 9 × 105 cells/mL. 5ALK-23 remained stable in fuel and detected both planktonic cells and biofilms. The validated 5ALK-23 BRE can be used for functionalization of biosensors for detection of fungi in fuel.

Highly catalytic nanoenzyme of covalent organic framework loaded starch- surface-enhanced Raman scattering/absorption bi-mode peptide as biosensor for ultratrace determination of cadmium.

High affinity peptides (PTs) have been used in nanoanalysis, but there are no reports which combine PTs with a liquid crystal (LC) covalent organic framework (COF) supported soluble starch (SS) catalytic amplification system as a biosensor recognition element. In this study, a new, highly sensitive and selective bi-mode molecular biosensor has been developed for the determination of cadmium ion (Cd2+). Specifically, a highly catalytic and stable COF supported SS nanosol catalyst was fabricated such that a nanocatalytic indicator reaction system for HAuCl4-sodium formate was established based on surface-enhanced Raman scattering (SERS). The Au nanoparticles produced exhibited a surface plasmon resonance (SPR) absorption peak at 535 nm and a SERS peak at 1,615 cm-1. Combining the nanocatalytic amplification indicator system with the specific PTs reaction permitted a sensitive and selective SERS/absorption bi-mode platform to be developed for the determination of cadmium in rice. The linear range for SERS determination was 0.025-0.95 nmol/L and the detection limit (DL) was 0.012 nmol/L.

Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9-RAD1-HUS1 (9-1-1) Complex.

Affinity pulldown is a powerful technique to discover novel interaction partners and verify a predicted physical association between two or more proteins. Pulldown assays capture a target protein fused with an affinity tag and analyze the complexed proteins. Here, we detail methods of pulldown assays for two high-affinity peptide fusion tags, Flag tag (DYKDDDDK) and hexahistidine tag (6xHis), to study protein-protein interactions of human NEIL1 glycosylase and the checkpoint protein complex RAD9-RAD1-HUS1 (9-1-1). We uncover unique interactions between 9-1-1 and NEIL1, which suggest a possible inhibitory role of the disordered, phosphorylated C-terminal region of RAD9 in regulating NEIL1 activity in base excision repair through lack of physical association of 9-1-1 and NEIL1.

High Potency Vasoactive Intestinal Peptide Receptor Antagonists Induce Durable Antileukemia/Tumor T Cell Memory in Mice with Once Daily Dosing

Introduction: Allogeneic bone marrow transplantation (allo-BMT) is curative for patients with myeloid leukemia (AML) through the activity of cytotoxic effector donor T cells, but high morbidity and mortality of allo-BMT limits the use of this therapy. A long-standing goal is the development of strategies that induce an autologous immune response to leukemic blasts by the patient's T cells. Vasoactive intestinal peptide (VIP) is a neuropeptide with pleiotropic functions, including modulation of immune and inflammatory responses. Previous studies demonstrated that VIP hybrid (VIPhyb), a peptide antagonist of VIP-signaling, induced T cell-dependent anti-leukemic activity in murine allotransplant models and promoted activation of mouse and human T cells (Li et al., 2016, Cancer Res). We developed a series of novel peptide VIP antagonists (VIP-ANT) based upon predicted binding affinity in silico to human VIP receptors VPAC1 and VPAC2. We tested the anti-cancer potential of these new VIP-ANT peptides in murine models of myeloid leukemia and mastocytoma, investigating the optimal dose and schedule of administration, and explored mechanisms underlying anti-tumor memory induced by VIP-ANT peptides. Methods: B6 (CD45.1, or albino) mice were injected intravenously with 1 x 106 C1498 cells (AML), and DBA/2j mice were injected subcutaneously with 5 x 104 P815 cells (myeloid mastocytoma). Leukemia-bearing mice were treated with subcutaneous injections of VIP-ANT or control scrambled peptides. Treatment started six days after injection of cells when C1498 leukemia cells were detectable in the blood, and luciferase+ P815 cells as tumors were palpable. A range of VIP-ANT doses was administered s.c. at 10, 30, or 100 μg. Treatment schedules tested included twice daily for 14 doses, once daily, once every other day, once every three days, and once weekly for 7 doses. Some mice received a single dose at day +6 following leukemia cell injection. Lymphocyte kinetics and leukemia cell burden were analyzed weekly through submandibular peripheral blood collections. Mice without evidence of AML/mastocytoma >60 days post-inoculation received a second injection of a two-fold higher dose of C1498 (2 × 106 cells) or P815 (1 × 105 cells). Results: We tested over 13 new VIP-ANT peptides. Daily or every other day (qod) administration of the novel VIP-ANT peptides to leukemia/tumor-bearing mice led to clearance of C1498 cells from blood or complete regression of P815 in 40 % of mice, leading to significantly improved survival over 60 days. Daily administration of 10 μg of novel VIP-ANT peptides for 7 days to leukemic mice prolonged survival(p<0.0001 VIP-ANT vs scramble, Fig 1A), with the fraction of mice that remained leukemia-free proportional to the predicted binding affinity of the VIP-ANT to human VPAC1 and VPAC2. With increased dose intensity and dose-density of ANT308, the most potent peptide, 40%-80% of mice survived to 70 days (median survival days (MSD) of 60 days) after ANT308 treatment compared with the survival of <20% in mice receiving scrambled peptide (MSD 36 days). Increasing the dose and schedule of ANT308 administration to 100 μg twice-daily led to 65% survival (MSD not reached) versus 40% with daily dosing, 30% with every-other-day dosing, and 0% survival with scrambled-sequence control peptide (MSD 33 days) (p<0.001, Fig 1B). Notably, the affinity of different VIP-ANT peptides to VPAC1 and VPAC2 VIP receptors significantly correlated with the survival of C1498-leukemic mice following prior treatment with the corresponding peptide. Prior treatment of leukemic mice with VIP-ANT induced protective immunological memory, rendering mice resistant to re-challenge with C1498 acute myeloid leukemia (80% survival) and P815 mastocytoma (60% survival) cells, compared with 0% survival (MSD 21 days) among immunologically naïve mice. Conclusion: Blocking VIP-signaling with high-affinity VIP-R peptide antagonists represents a novel pharmacological approach to enhance adaptive T cell responses to leukemia/myeloid sarcoma-associated antigens in murine leukemia/tumor models. We are continuing PK and toxicology studies with the lead VIP-ANT peptides to support the development of phase 1 clinical trials for patients with relapsed AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance.

SUMMARYPrevention or amelioration of declining β cell mass is a potential strategy to cure diabetes. Here, we report the pathways utilized by β cells to robustly replicate in response to acute insulin resistance induced by S961, a pharmacological insulin receptor antagonist. Interestingly, pathways that include CENP-A and the transcription factor E2F1 that are independent of insulin signaling and its substrates appeared to mediate S961-induced β cell multiplication. Consistently, pharmacological inhibition of E2F1 blocks β-cell proliferation in S961-injected mice. Serum from S961-treated mice recapitulates replication of β cells in mouse and human islets in an E2F1-dependent manner. Co-culture of islets with adipocytes isolated from S961-treated mice enables β cells to duplicate, while E2F1 inhibition limits their growth even in the presence of adipocytes. These data suggest insulin resistance-induced proliferative signals from adipocytes activate E2F1, a potential therapeutic target, to promote β cell compensation.

Enzymatic processing and MHC loading 3Molecular mechanisms of chaperone-mediated MHC-I peptide loading

Peptide loading of MHC-I molecules occurs as a result of dynamic protein folding and assembly processes involving not only the MHC-I heavy chain, the light chain, beta 2-m, and antigenic peptide, but also the components of the peptide loading complex (PLC), including tapasin, as well as the PLC-independent chaperone, TAP binding protein, related (TAPBPR). Structural studies of tapasin and TAPBPR reveal that these, like many other proteins, contain both well-ordered domains as well as regions of disorder. To examine the contributions of disordered regions of both MHC-I molecules and of TAPBPR, we have examined X-ray structures of MHC-I molecules assembled with disulfide linked truncated peptides, of TAPBPR/MHC-I complexes containing truncated peptides, and of nanobody/TAPBPR complexes. Different disordered regions of TAPBPR rigidify on interaction with MHC-I or with specific nanobodies. Conversely, the peptide binding groove of MHC-I structurally rearranges on interaction with TAPBPR. These observations are consistent with solution nuclear magnetic resonance (NMR) spectroscopy data examining the interaction of TAPBPR with isotope-labeled peptide-MHC-I complexes. The dynamic NMR data reveal a transient pMHC-I/TAPBPR intermediate that controls the selection of high-affinity peptides. We propose a model of negative allosteric coupling that involves the entire peptide binding groove. Such a model, based primarily on observations of TAPBPR should be applicable to tapasin in the PLC as well.

A 3D Peptide/[60]Fullerene Hybrid for Multivalent Recognition.

Fully substituted peptide/[60]fullerene hexakis-adducts offer an excellent opportunity for multivalent protein recognition. In contrast to monofunctionalized fullerene hybrids, peptide/[60]fullerene hexakis-adducts display multiple copies of a peptide in close spatial proximity and in the three dimensions of space. High affinity peptide binders for almost any target can be currently identified by in vitro evolution techniques, often providing synthetically simpler alternatives to natural ligands. However, despite the potential of peptide/[60]fullerene hexakis-adducts, these promising conjugates have not been reported to date. Here we present a synthetic strategy for the construction of 3D multivalent hybrids that are able to bind with high affinity the E-selectin. The here synthesized fully substituted peptide/[60]fullerene hybrids and their multivalent recognition of natural receptors constitute a proof of principle for their future application as functional biocompatible materials.

A 3D Peptide/[60]Fullerene Hybrid for Multivalent Recognition

AbstractFully substituted peptide/[60]fullerene hexakis‐adducts offer an excellent opportunity for multivalent protein recognition. In contrast to monofunctionalized fullerene hybrids, peptide/[60]fullerene hexakis‐adducts display multiple copies of a peptide in close spatial proximity and in the three dimensions of space. High affinity peptide binders for almost any target can be currently identified by in vitro evolution techniques, often providing synthetically simpler alternatives to natural ligands. However, despite the potential of peptide/[60]fullerene hexakis‐adducts, these promising conjugates have not been reported to date. Here we present a synthetic strategy for the construction of 3D multivalent hybrids that are able to bind with high affinity the E‐selectin. The here synthesized fully substituted peptide/[60]fullerene hybrids and their multivalent recognition of natural receptors constitute a proof of principle for their future application as functional biocompatible materials.

Site-directed dual bioprobes inducing single-step nano-sandwich assay for the detection of cardiac troponin I.

Bioreceptor functionalized metallic nano-colloids have been identified as effective nanobioprobes to realize the detection of an analyte based on a common phenomenon of salt-induced aggregation. In marked contrast to this, we describe a nano-sandwich assay integrating the novel match-pair of aptamer and peptide functionalized gold nanoparticles. The site-directed biomolecular interaction of high affinity aptamer and peptide bioreceptors directed towards distinct sites of cardiac biomarker troponin I; this was found to form a nano-sandwich assay in a peculiar manner. The gold nanoconjugates interact with specific and distant regions of troponin I to result in collision of probes upon target identification. In the presence of TnI, both nanobioprobes bind at their respective sites forming a nano-sandwich pair providing a visual color change from red to blue. Thus, the presence of target TnI itself causes instant agglomeration in just a single-step without addition of any external aggregator. The assay imparts 100% specificity and 90% sensitivity in a dynamic concentration range of 0.1-500ng/mL troponin I with detection limit as low as 0.084ng/mL. The applicability of the assay has been validated in clinical samples of acute myocardial infarction patients thus establishing a promising point-of-care detection of TnI.

131I-Caerin 1.1 and 131I-Caerin 1.9 for the treatment of non-small-cell lung cancer.

ObjectiveTo investigate the effect of the 131I-labeled high-affinity peptides Caerin 1.1 and Caerin 1.9 for the treatment of A549 human NSCLC cells.Methods ① 3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and plate clone formation assays were performed to confirm the in vitro anti-tumor activity of Caerin 1.1 and Caerin 1.9. ② Chloramine-T was used to label Caerin 1.1 and Caerin 1.9 with 131I, and the Cell Counting Kit 8 assay was performed to analyze the inhibitory effect of unlabeled Caerin 1.1, unlabeled Caerin 1.9, 131I-labeled Caerin 1.1, and 131I-labeled Caerin 1.9 on the proliferation of NSCLC cells. An A549 NSCLC nude mouse model was established to investigate the in vivo anti-tumor activity of unlabeled Caerin 1.1, unlabeled Caerin 1.9, 131I-labeled Caerin 1.1, and 131I-labeled Caerin 1.9.Results ① Caerin 1.1 and Caerin 1.9 inhibited the proliferation of NSCLC cells in vitro in a concentration-dependent manner. The half-maximal inhibitory concentration was 16.26 µg/ml and 17.46 µg/ml, respectively, with no significant intergroup difference (P>0.05). ② 131I-labeled Caerin 1.1 and 131I-labeled Caerin 1.9 were equally effective and were superior to their unlabeled versions in their ability to inhibit the proliferation and growth of NSCLC cells (P>0.05).Conclusions 131I-labeled Caerin 1.1 and 131I-labeled Caerin 1.9 inhibit the proliferation and growth of NSCLC cells and may become potential treatments for NSCLC.

Affinity selection-mass spectrometry in the discovery of anti-SARS-CoV-2 compounds.

Small molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), which is the cause of the COVID‐19 pandemic. To expedite the discovery of lead compounds for development, assays have been developed based on affinity selection‐mass spectrometry (AS‐MS), which enables the rapid screening of mixtures such as combinatorial libraries and extracts of botanicals or other sources of natural products. AS‐MS assays have been used to find ligands to the SARS‐CoV‐2 spike protein for inhibition of cell entry as well as to the 3‐chymotrypsin‐like cysteine protease (3CLpro) and the RNA‐dependent RNA polymerase complex constituent Nsp9, which are targets for inhibition of viral replication. The AS‐MS approach of magnetic microbead affinity selection screening has been used to discover high‐affinity peptide ligands to the spike protein as well as the hemp cannabinoids cannabidiolic acid and cannabigerolic acid, which can prevent cell infection by SARS‐CoV‐2. Another AS‐MS method, native mass spectrometry, has been used to discover that the flavonoids baicalein, scutellarein, and ganhuangenin, can inhibit the SARS‐CoV‐2 protease 3CLpro. Native mass spectrometry has also been used to find an ent‐kaurane natural product, oridonin, that can bind to the viral protein Nsp9 and interfere with RNA replication. These natural lead compounds are under investigation for the development of therapeutic agents to prevent or treat SARS‐CoV‐2 infection.

The Screening of Therapeutic Peptides for Anti-Inflammation through Phage Display Technology.

For the treatment of inflammatory illnesses such as rheumatoid arthritis and carditis, as well as cancer, several anti-inflammatory medications have been created over the years to lower the concentrations of inflammatory mediators in the body. Peptides are a class of medication with the advantages of weak immunogenicity and strong activity, and the phage display technique is an effective method for screening various therapeutic peptides, with a high affinity and selectivity, including anti-inflammation peptides. It enables the selection of high-affinity target-binding peptides from a complex pool of billions of peptides displayed on phages in a combinatorial library. In this review, we will discuss the regular process of using phage display technology to screen therapeutic peptides, and the peptides screened for anti-inflammation properties in recent years according to the target. We will describe how these peptides were screened and how they worked in vitro and in vivo. We will also discuss the current challenges and future outlook of using phage display to obtain anti-inflammatory therapeutic peptides.

Peptide-to-Small Molecule: A Pharmacophore-Guided Small Molecule Lead Generation Strategy from High-Affinity Macrocyclic Peptides.

Recent technological innovations have led to the development of methods for the rapid identification of high-affinity macrocyclic peptides for a wide range of targets; however, it is still challenging to achieve the desired activity and membrane permeability at the same time. Here, we propose a novel small molecule lead discovery strategy, ″Peptide-to-Small Molecule″, which is a combination of rapid identification of high-affinity macrocyclic peptides via peptide display screening followed by pharmacophore-guided de novo design of small molecules, and demonstrate the applicability using nicotinamide N-methyltransferase (NNMT) as a target. Affinity selection by peptide display technology identified macrocyclic peptide 1 that exhibited good enzymatic inhibitory activity but no cell-based activity. Thereafter, a peptide pharmacophore-guided de novo design and further structure-based optimization resulted in highly potent and cell-active small molecule 14 (cell-free IC50 = 0.0011 μM, cell-based IC50 = 0.40 μM), indicating that this strategy could be a new option for drug discovery.

Molecular dynamics simulations of cRGD-conjugated PEGylated TiO2 nanoparticles for targeted photodynamic therapy

The conjugation of high-affinity cRGD-containing peptides is a promising approach in nanomedicine to efficiently reduce off-targeting effects and enhance the cellular uptake by integrin-overexpressing tumor cells. Herein we utilize atomistic molecular dynamics simulations to evaluate key structural–functional parameters of these targeting ligands for an effective binding activity towards αVβ3 integrins. An increasing number of cRGD ligands is conjugated to PEG chains grafted to highly curved TiO2 nanoparticles to unveil the impact of cRGD density on the ligand’s presentation, stability, and conformation in an explicit aqueous environment. We find that a low density leads to an optimal spatial presentation of cRGD ligands out of the “stealth” PEGylated layer around the nanosystem, favoring a straight upward orientation and spaced distribution of the targeting ligands in the bulk-water phase. On the contrary, high densities favor over-clustering of cRGD ligands, driven by a concerted mechanism of enhanced ligand–ligand interactions and reduced water accessibility over the ligand’s molecular surface. These findings strongly suggest that the ligand density modulation is a key factor in the design of cRGD-targeting nanodevices to maximize their binding efficiency into over-expressed αVβ3 integrin receptors.