Conversion of Low‐Affinity Peptides to High‐Affinity Peptide Binders by Using a β‐Hairpin Scaffold‐Assisted Approach

Affinity maturation of protein‐targeting peptides is generally accomplished by homo‐ or heterodimerization of known peptides. However, applying a heterodimerization approach is difficult because it is not clear a priori what length or type of linker is required for cooperative binding to a target. Thus, an efficient and simple affinity maturation method for converting low‐affinity peptides into high‐affinity peptides would clearly be advantageous for advancing peptide‐based therapeutics. Here, we describe the development of a novel affinity maturation method based on a robust β‐hairpin scaffold and combinatorial phage‐display technology. With this strategy, we were able to increase the affinity of existing peptides by more than four orders of magnitude. Taken together, our data demonstrate that this scaffold‐assisted approach is highly efficient and effective in generating high‐affinity peptides from their low‐affinity counterparts.     

Arginine mimetics using α-guanidino acids: introduction of functional groups and stereochemistry adjacent to recognition guanidiniums in peptides

Arginine residues are broadly employed for specific biomolecular recognition, including in protein-protein, protein-DNA, and protein-RNA interactions. Arginine recognition commonly exploits the potential for bidentate electrostatic and hydrogen-bonding interactions. However, in arginine residues, the guanidinium functional group is located at the terminus of a flexible hydrocarbon side chain, which lacks the functionality to contribute to specific arginine-mediated recognition and may entropically disfavor binding. In order to enhance the potential for specificity and affinity in arginine-mediated molecular recognition, we have developed an approach to the synthesis of peptides that incorporates an α-guanidino acid as a novel arginine mimetic. α-Guanidino acids, derived from α-amino acids, with guanidinylation of the amino group, were incorporated stereospecifically into peptides on solid phase via coupling of an Fmoc amino acid to diaminopropionic acid (Dap), Fmoc deprotection, guanidinylation of the amine on solid phase, and deprotection, generating a peptide containing an α-functionalized arginine mimetic. This approach was examined by incorporating arginine mimetics into ligands for the Src, Grb, and Crk SH3 domains at the site of the key recognition arginine. Protein binding was examined for peptides containing guanidino acids derived from Gly, L-Val, L-Phe, L-Trp, D-Val, D-Phe, and D-Trp. We demonstrate that paralogue specificity and target site affinity may be modulated with the use of α-guanidino acid-derived arginine mimetics, generating peptides that exhibit enhanced Src specificity by selection against Grb and peptides that reverse the specificity of the native peptide ligand, with enhancements in Src target specificity of up to 15-fold (1.6 kcal mol(-1)).     

The Role of the VEGF-C/VEGFRs Axis in Tumor Progression and Therapy

Vascular endothelial growth factor C (VEGF-C) has been identified as a multifaceted factor participating in the regulation of tumor angiogenesis and lymphangiogenesis. VEGF-C is not only expressed in endothelial cells, but also in tumor cells. VEGF-C signaling is important for progression of various cancer types through both VEGF receptor-2 (VEGFR-2) and VEGF receptor-3 (VEGFR-3). Likewise, both receptors are expressed mainly on endothelial cells, but also expressed in tumor cells. The dimeric VEGF-C undergoes a series of proteolytic cleavage steps that increase the protein binding affinity to VEGFR-3; however, only complete processing, removing both the N- and C-terminal propeptides, yields mature VEGF-C that can bind to VEGFR-2. The processed VEGF-C can bind and activate VEGFR-3 homodimers and VEGFR-2/VEGFR-3 heterodimers to elicit biological responses. High levels of VEGF-C expression and VEGF-C/VEGFRs signaling correlate significantly with poorer prognosis in a variety of malignancies. Therefore, the development of new drugs that selectively target the VEGF-C/VEGFRs axis seems to be an effective means to potentiate anti-tumor therapies in the future.     

Urea-Peptide Hybrids as VEGF-A165/NRP-1 Complex Inhibitors with Improved Receptor Affinity and Biological Properties

Neuropilin-1 (NRP-1), the major co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2), may also independently act with VEGF-A₁₆₅ to stimulate tumour growth and metastasis. Therefore, there is great interest in compounds that can block VEGF-A165/NRP-1 interaction. Peptidomimetic type inhibitors represent a promising strategy in the treatment of NRP-1-related disorders. Here, we present the synthesis, affinity, enzymatic stability, molecular modeling and in vitro binding evaluation of the branched urea–peptide hybrids, based on our previously reported Lys(hArg)-Dab-Oic-Arg active sequence, where the Lys(hArg) branching has been modified by introducing urea units to replace the peptide bond at various positions. One of the resulting hybrids increased the affinity of the compound for NRP-1 more than 10-fold, while simultaneously improving resistance for proteolytic stability in serum. In addition, ligand binding to NRP-1 induced rapid protein stock exocytotic trafficking to the plasma membrane in breast cancer cells. Examined properties characterize this compound as a good candidate for further development of VEGF₁₆₅/NRP-1 inhibitors.     

A powerful combinatorial screen to identify high-affinity terbium(III)-binding peptides

Lanthanide-binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb(3+) ions and intense luminescence properties. This methodology utilizes solid-phase split-and-pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two-step screening procedure. The initial screen avoids the interference caused by on-bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aqueous solution. Employment of this methodology on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140-fold increase in affinity (57 nM dissociation constant, K(D)) over previously reported lanthanide-binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissociation constant for Tb(3+) ions.Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.     

Bicyclic peptide antagonists derived from genetically encoded combinatorial libraries

Ligands based on bicyclic peptides can combine favourable properties of antibodies (good binding affinity and target specificity) and small molecule ligands (stability, access to chemical synthesis, diffusion properties) and might be suitable molecular structures for the development of therapeutics. By using a combinatorial methodology based on phage display and a chemical cyclisation reaction, we are generating bicyclic peptide antagonists of protein targets with therapeutic applications in mind.     

Two-Helix Supramolecular Proteomimetic Binders Assembled via PNA-Assisted Disulfide Crosslinking

Peptidic motifs folded in a defined conformation are able to inhibit protein-protein interactions (PPIs) covering large interfaces and as such they are biomedical molecules of interest. Mimicry of such natural structures with synthetically tractable constructs often requires complex scaffolding and extensive optimization to preserve the fidelity of binding to the target. Here, we present a novel proteomimetic strategy based on a 2-helix binding motif that is brought together by hybridization of peptide nucleic acids (PNA) and stabilized by a rationally positioned intermolecular disulfide crosslink. Using a solid phase synthesis approach (SPPS), the building blocks are easily accessible and such supramolecular peptide-PNA helical hybrids could be further coiled using precise templated chemistry. The elaboration of the structural design afforded high affinity SARS CoV-2 RBD (receptor binding domain) binders without interference with the underlying peptide sequence, creating a basis for a new architecture of supramolecular proteomimetics.     

In Silico Design of MDM2-Targeting Peptides from a Naturally Occurring Constrained Peptide

Naturally occurring constrained peptides are frequently used as scaffolds for bioactive peptide grating due to their high stability. Here, we used in silico methods to design several constrained peptides comprising a scorpion toxin scaffold, a MDM2 binding epitope, and a cluster of positively charged residues. The designed peptides displayed varied binding affinity to MDM2 despite differing by only one or two residues. One of the peptides, SC426, had nanomolar binding affinity (K_D=6.6±2.6 nm ) to MDM2, and exhibited stronger inhibitory activity on the proliferation of HCT116 cells (p53-wild type) and SW480 cells (p53-mutant) than that of nutlin-3a. Binding mode analysis of the designed peptide at MDM2 suggests that the conserved “FWL” epitope was buried in the hydrophobic binding pocket, and the residues located at the periphery of the binding site contributed to the high binding affinity of SC426. Overall, in silico design of miniproteins with therapeutic potential through epitope grafting to the naturally occurring constrained peptide is an effective strategy.     

Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands

We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[E-LYLAYPAH-K] featured a K_D of 1.75 μM for YAP and 0.68 μM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.     

Iterative Nonproteinogenic Residue Incorporation Yields α/β‐Peptides with a Helix–Loop–Helix Tertiary Structure and High Affinity for VEGF

Inhibition of specific protein–protein interactions is attractive for a range of therapeutic applications, but the large and irregularly shaped contact surfaces involved in many such interactions make it challenging to design synthetic antagonists. Here, we describe the development of backbone‐modified peptides containing both α‐ and β‐amino acid residues (α/β‐peptides) that target the receptor‐binding surface of vascular endothelial growth factor (VEGF). Our approach is based on the Z‐domain, which adopts a three‐helix bundle tertiary structure. We show how a two‐helix “mini‐Z‐domain” can be modified to contain β and other nonproteinogenic residues while retaining the target‐binding epitope by using iterative unnatural residue incorporation. The resulting α/β‐peptides are less susceptible to proteolysis than is their parent α‐peptide, and some of these α/β‐peptides match the full‐length Z‐domain in terms of affinity for receptor‐recognition surfaces on the VEGF homodimer.     

Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A₁₆₅ region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B₁₆₇ derived peptides were more effective than VEGF-A₁₆₅ peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.     

An in vitro Assay to Measure Targeted Drug Delivery to Bone Mineral

Targeted delivery of drugs to their site of action is a promising strategy to decrease adverse effects and enhance efficacy, but successful applications of this strategy have been scarce. Human bone is a tissue with unique properties due to its high hydroxyapatite mineral content. However, with the exception of bisphosphonates, bone mineral has not been targeted in a successful clinical application of drugs that act on bone, such as anti‐resorptive or bone anabolic agents. Herein we present an NMR‐based in vitro assay to measure binding affinities of small molecules to hydroxyapatite (HAP) or bone powder. Binding was shown to be specific and competitive, and the assay can be carried out in a direct binding format or in competition mode. A selection of clinically relevant bisphosphonates was ranked by their binding affinity for HAP. The binding affinity decreases in the order: pamidronate > alendronate > zoledronate > risedronate > ibandronate. The differences in binding affinities span a factor of 2.1 between pamidronate and ibandronate, consistent with previous studies. The rank order is very similar with bone powder, although the binding capacity of bone powder is smaller and binding kinetics are slower. A zoledronate derivative that lacks the central hydroxy group binds to HAP with 2.3‐fold weaker affinity than zoledronate itself. Any small molecule can be analyzed for its binding to HAP or bone powder, and the binding of common bone‐staining agents such as alizarin and its derivatives was confirmed in the new assay. This assay supports a strategy for targeted delivery of drugs to bone by attaching a bone‐affinity tag to the active drug substance.     

Resin-bound aminofluorescein for C-terminal labeling of peptides: high-affinity polarization probes binding to polyproline-specific GYF domains

A polymer support for the solid-phase synthesis of C-terminally labeled carboxylic acids has been developed. Fluorophore-labeled peptides were constructed directly on the amino group of resin-bound aminofluorescein. Fmoc-protected aminofluorescein was coupled onto tritylpolystyrene, and the free phenolic hydroxyl positions of the fluorescein were blocked with suitable protecting groups. The mode of attachment was analyzed and found to be selective for the phenoxy ether linkage. The conditions for peptide synthesis on the labeling resin were investigated, and a small library of C-terminally labeled peptides was prepared. The fluorescence quantum yields of C-terminally labeled peptides were determined and indicated the suitability of the compounds for imaging and binding experiments. The obtained peptides were therefore investigated as fluorescence polarization probes. Two different proline-rich binding domains of the GYF family-CD2BP2 and PERQ2-were targeted by peptides labeled either C- or N-terminally. Reversible binding constants were determined by fluorescence polarization measurements and were verified by competition experiments with the corresponding unlabeled peptide. As a second control, the binding constants were measured by NMR titration experiments, recording the HSQC NMR spectra of (15)N-labeled proteins in the presence of the peptide polarization probes. Ligands with higher affinities than all others known previously were identified for both GYF domains. The competition assay with the developed fluorescent probe has a high statistical reliability and can thus be used for screening of GYF domain inhibitors.     

Affinity and Specificity for Binding to Glycosaminoglycans Can Be Tuned by Adapting Peptide Length and Sequence

Although glycosaminoglycan (GAG)–protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.     

Crystalline selenite substituted carbonated hydroxyapatite nanorods: Synthesis,characterization, evaluation of bioactivity and cytotoxicity

Synthesis of pure and selenite substituted hydroxyapatites (HAP and Se‐HAP with 0.02–0.10 Se) by sol–gel method and evaluation of their morphological features, nature of functional groups, phase purity, in vitro bioactivity and cytotoxicity are addressed. Spectral studies confirm the incorporation of selenite into the HAP lattice, accompanied by an increase in CO₃^2? content to maintain the charge imbalance following replacement of P⁵⁺ with Se⁴⁺, thus making the formation of selenite substituted carbonated HAP. Selenite substitution in the HAP lattice has led to a higher crystallinity and increased the crystallite size. The morphology of HAP is changed from sphere to rod‐like structure upon substitution by selenite and the size of the rod is increased with an increase in the selenite content. Among the Se‐HAPs, a better in vitro bioactivity and cell viability are observed for 0.02 Se‐HAP and 0.04 Se‐HAP while the trend is reversed when the extent of selenite substitution becomes higher.     

In Silico Discovery of Potential VEGFR-2 Inhibitors from Natural Derivatives for Anti-Angiogenesis Therapy

Angiogenesis is the growth of new capillaries from existing blood vessels that supply oxygen and nutrients and provide gateways for immune surveillance. Abnormal vessel growth in term of excessive angiogenesis is a hallmark of cancer, inflammatory and eye diseases. VEGFR-2 (vascular endothelial growth factor receptor 2) dominating the process of angiogenesis has led to approval of therapeutic inhibitors and is becoming a promising target for anti-angiogenic drugs. Notwithstanding these successes, the clinical use of current VEGFR-2 blockers is more challenging than anticipated. Taking axitinib as a reference drug, in our study we found three potent VEGFR-2 inhibitors (ZINC08254217, ZINC08254138, and ZINC03838680) from natural derivatives. Each of the three inhibitors acquired a better grid score than axitinib (−62.11) when docked to VEGFR-2. Molecular dynamics simulations demonstrated that ZINC08254217– and ZINC08254138–VEGFR-2 complexes were more stable than axitinib. Similar to bind free energy for axitinib (−54.68 kcal/mol), such for ZINC03838680, ZINC08254217, and ZINC08254138 was −49.37, −43.32, and −32.73 kcal/mol respectively. These results suggested these three compounds could be candidate drugs against angiogenesis, with comparable VEGFR-2 binding affinity of axitinib. Hence findings in our study are able to provide valuable information on discovery of effective anti-angiogenesis therapy.     

Landscape phages and their fusion proteins targeted to breast cancer cells

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific 'fusion pVIII proteins' (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage-cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1.     

Trastuzumab-Peptide Interactions: Mechanism and Application in Structure-Based Ligand Design

Understanding of protein-ligand interactions and its influences on protein stability is necessary in the research on all biological processes and correlative applications, for instance, the appropriate affinity ligand design for the purification of bio-drugs. In this study, computational methods were applied to identify binding site interaction details between trastuzumab and its natural receptor. Trastuzumab is an approved antibody used in the treatment of human breast cancer for patients whose tumors overexpress the HER2 (human epidermal growth factor receptor 2) protein. However, rational design of affinity ligands to keep the stability of protein during the binding process is still a challenge. Herein, molecular simulations and quantum mechanics were used on protein-ligand interaction analysis and protein ligand design. We analyzed the structure of the HER2-trastuzumab complex by molecular dynamics (MD) simulations. The interaction energies of the mutated peptides indicate that trastuzumab binds to ligand through electrostatic and hydrophobic interactions. Quantitative investigation of interactions shows that electrostatic interactions play the most important role in the binding of the peptide ligand. Prime/MM-GBSA calculations were carried out to predict the binding affinity of the designed peptide ligands. A high binding affinity and specificity peptide ligand is designed rationally with equivalent interaction energy to the wild-type octadecapeptide. The results offer new insights into affinity ligand design.     

Increased Valency Improves Inhibitory Activity of Peptides Targeting Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a clinically validated target for treating hypercholesterolemia. Peptide-based PCSK9 inhibitors have attracted pharmaceutical interest, but the effect of multivalency on bioactivity is poorly understood. Here we designed bivalent and tetravalent dendrimers, decorated with the PCSK9 inhibitory peptides Pep2-8[RRG] or P9-38, to study relationships between peptide binding affinity, peptide valency, and PCSK9 inhibition. Increased valency resulted in improved PCSK9 inhibition for both peptides, with activity improvements of up to 100-fold achieved for the P9-38-decorated dendrimers compared to monomeric P9-38 in in vitro competition binding assays. Furthermore, the P9-38-decorated dendrimers showed improved potency at restoring functional low-density lipoprotein (LDL) receptor levels and internalizing LDL in the presence of PCSK9, demonstrating significant cell-based activity at picomolar concentrations. This study demonstrates the potential of increasing valency as a strategy for increasing the efficacy of peptide-based PCSK9 therapeutics.