Peptides

In this study, we developed a new peptide motif called β-strap (strap = strand + cap) used to fold β-hairpins of varying length. β-Straps are mean to be short sequences (4 to 8 a-amino acids) forming β-sheets using a judicious combination of non-covalent interactions (NCI) to overcome the entropic penalty inherent to long loop closure. Among those, we proved that a couple of CH-π / NH-π interactions from a tryptophan zipper motif were critical to create a stable packing of the structure. To optimize these interactions, we incorporated unnatural tryptophan derivatives having functionalized indole side chains. Finally, the innate ability of the β-strap to bring β-stand in close contact was exploited to promote macrocyclization of long coiled peptides (up to 16 residues).
Then, we studied a more complex β-hairpin loop mimics found at the apex of monoclonal antibodies (mAb) complementary determining region 3 (CDR-H3). Using a set of bioinformatics tools, a search of PDB crystal structures revealed that a large set of mAb crystals possess a β-bulge, located at the edge of CDR-H3 loops. A cluster analysis revealed it has an impressive adaptability towards different H3-loop sizes and conformations. In order to evaluate its function in antibodies, we synthesized several β-hairpin models bearing a prototypical β-bulge. By combining short β-straps and the β-bulge, we were able to design β-hairpin peptides mimic of mAb with a variety of lengths and rigidity while retaining a high degree of folding. Starting from pembrolizumab, the most outstanding blocker of the PD-1/PD-L1 checkpoint currently available in clinic, we scoped ~30 CDR-H3 mAb mimics (H3 loop). As a result, several novel β-hairpin peptide inhibitors of the PD-1/PD-L1 pathway were identified (IC50 <0.3 μM).

Matrix Metalloproteinase-13 (MMP-13) belongs to a large family of proteolytic enzymes which are characterized by their ability to degrade the extracellular matrix components. MMP-13 appears to have a critical role in tumor invasion and metastasis. In this study, several fluorogenic probes specific for MMP-13 were designed and characterized. These synthesized probes could be modified with chelators to be applied for imaging MMP-13 in breast cancer and/or multiple myeloma models. The activity and selectivity of MMP-13 and other MMPs against these probes were studied through two approaches. It was found that these probes were cleaved by all MMPs, but MMP-13 showed the highest activity and selectivity towards these peptides.

The misfolding of native, cellular prion protein (PrPc) to a conformationally altered pathogenic isoform, designated scrapie PrPsc, is the main molecular process involved in the pathogenesis of prion diseases. Prion diseases are marked by the accumulation of conformationally modified forms of cellular prion protein. An N-terminal portion of the prion protein, PrP (106-128), is a 23-residue peptide fragment and is characterized by an amphipathic structure with two domains: a hydrophilic N-terminal domain and a hydrophobic C-terminal domain. In this study, the aggregation characteristics of the PrP (106-128) peptide were investigated using a combination of biophysical approaches. We investigated the effect of different factors including concentrations, pH, and metal ions, on the aggregation of the peptide. Our results demonstrated that the peptide steadily aggregates at concentrations higher than 25 M. The aggregation propensity and fibril formation is higher at pH 7.4 and pH 8.1, and the aggregation is inhibited at pH lower than 6. Furthermore, our results indicate that the Cu2+ has much less effect on the peptide amyloidogenesis, while Zn2+ has a significant influence on the PrP (106-128) amyloidogenesis. We further presented a systematic analysis of the impact of phospholipid liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-racglycerol) (POPG) in the absence or presence of cholesterol, on the amyloidogenesis of PrP (106-128). The results showed that POPC vesicles does not significantly influence the aggregation kinetics of the peptide. However, the anionic lipid POPG delays the aggregation in a concentration-dependent manner, whereas the addition of POPG with the cholesterol shows fast kinetics of fibrillization, thus reducing the lag time of the aggregation kinetics. We also monitored the effect of cholesterol and its derivatives including cholesterol-SO4 and DC-cholesterol on PrP (106-128) amyloidogenesis. Our results showed that the cholesterol inhibits the peptide aggregation and delays the formation of fibrils in a concentration-dependent manner. Cholesterol-SO4 dramatically facilitates the aggregation at high concentrations but has the potential to slow down the fibrillization at low concentrations, whereas cationic DC-cholesterol vesicles can effectively inhibit peptide fibril formation at high concentrations.

Collagen is the major structural scaffold in the body and serves as barrier between tissues, and thus its turnover is tightly regulated. Collagen triple-helical structure renders it resistant to general proteolysis. Several proteases are capable of cleaving the triplehelical regions of collagen, including several mammalian matrix metalloproteinases (MMPs) and bacterial collagenases. MMP-mediated collagenolysis is associated with numerous diseases and some bacterial collagenases have found clinical application use due to their efficiency in the hydrolysis of the collagen triple-helix. A selective Förster resonance energy transfer triple-helical peptide (fTHP) probe for monitoring the activity of Clostridial collagenase has been developed. The fTHP [sequence: Gly-mep-Flp-(Glyvi Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] was stable at 37 °C and was efficiently hydrolyzed by bacterial collagenase (kcat/KM = 25,000 s -1 M-1) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The bacterial collagenase fTHP assay can be utilized in applications where specific activity towards triple-helical collagen needs to be evaluated, such as isolation of cells from various tissues. An fTHP scaffold was also utilized to evaluate the sequence preferences of eight MMPs. Residues spanning from P3 to P11 investigated using a positional scanning synthetic combinatorial library. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. The results of this study show that the P10 11 substrate play an important role in the collagenase-substrate interactions and that modifying these residues can drastically affect the affinity of MMPs towards THP substrates. The identified sequence preferences of MMPs will enable the design of selective triple-helical MMP probes that could be used for monitoring in vivo enzyme activity and enzyme-facilitated drug delivery.

Cone snails are carnivorous marine mollusks, utilizing their neuropeptide-rich venom for prey capture. The venom of Conus brunneus, a wide-spread Eastern Pacific vermivore, has not been extensively studied. In the current work, peptides from the dissected venom were characterized and tested using preliminary bioassays. Six peptides (A-F) were isolated and tested. Three peptide identities were determined by comparison with previously reported data: bru9a (A), bru3a (F), and an a-conotoxin (E). Preliminary screening in a stroke-related model of induced glutamate excitotoxicity in primary neuronal cells and PC12 cell cultures indicated potential neuroprotective activity of peptide fractions A, D, and F. Further testing is necessary to determine and verify structure, activity, target, and mechanism of action of the promising peptides from C. brunneus, which may prove effective neuropharmacological agents to treat stroke.

With antimicrobial resistance to current drugs steadily rising, the development of new antibiotics with novel mechanisms of action has become an imperative. The majority of life-threatening infections worldwide are caused by "ESKAPE" pathogens which are encountered in more than 40% of hospital-acquired infections, and are resistant to the majority of commonly used antibiotics. Naturally occurring cyclic depsipeptides, microbial secondary metabolites that contain one or more ester bonds in addition to amide bonds, have emerged as an important source of pharmacologically active compounds or lead structures for the development of novel antibiotics. Some of those peptides are either already marketed (daptomycin) or in advanced stages of clinical development (ramoplanin). Structurally simple, yet potent, fusaricidin/LI-F and lysobactin families of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents capable of overco ming infections caused by multidrug-resistant bacteria. These natural products exhibit potent antimicrobial activity against a variety of clinically relevant fungi and Gram-positive bacteria. Therefore, access to these classes of natural products and their synthetic analogs, combined with elucidation of their mode of action represent important initial steps toward full exploitation of their antmicrobial potential. This dissertation describes a general approach toward the solid-phase synthesis of fusaricidin/LI-F and lysobactin analogs and an extensive structure-activity relationship (SAR) study. We have devised a simple and robust preparation strategy based on standard Fmoc solid-phase peptide synthesis protocols.

Cell penetrating peptides (CPPs) have drawn the attention of researchers due to their ability to internalize large cargos into cells including cancer cells. The mechanism(s) with which the peptides enter the cell, however, is/are not clear and full of controversy. The peptide conformations and their microenvironment in live cells had been unknown until the development of a technique developed in our lab. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 to 967 cm-1, thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals.

Cone snails are predatory marine gastropods that use venom for means of predation and defense. This venom is a complex mixture of conopeptides that selectivity binds to ion channels and receptors, giving them a wide range of potential pharmaceutical applications. Conus brunneus is a wide spread Eastern Pacific cone snail species that preys upon worms (vermivorous). Vermivorous cone snails have developed very specific biochemical strategies for the immobilization of their prey and their venom has not been extensively studied to date. The main objective of this dissertation is the characterization of novel conopeptides isolated from Conus brunneus. Chapter 1 is an introduction and background on cone snails and conopeptides. Chapter 2 details the isolation and characterization of a novel P-superfamily conotoxin. Chapter 3 presents the 3D solution structure of the novel P-superfamily conotoxin. Chapter 4 details the isolation and characterization of two novel M-superfamily conotoxins. Chapter 5 covers the use of nano-NMR to characterize a novel P-superfamily conotoxin using nanomole quantities of sample. Chapter 6 is a reprint of a paper published in the Journal of the American Chemical Society in which we combined and implemented techniques developed in the previous chapters to report the presence of D-(Sd(B-Hydroxyvaline in a polypeptide chain. This dissertation contains the first reported work of a P-superfamily structure obtained directly from the crude venom therefore accurately representing native post-translational modifications. In this paper, crude cone snail venom was characterized by: high performance liquid chromatography, nuclear magnetic resonance spectroscopy, nanonuclear magnetic resonance spectroscopy, mass spectrometry, amino acid analysis, Edman degradation sequencing, and preliminary bioassays.