Discovery and Characterization of MaK: A Novel Knottin Antimicrobial Peptide from Monochamus alternatus.

Knottin-type antimicrobial peptides possess exceptional attributes, such as high efficacy, low vulnerability to drug resistance, minimal toxicity, and precise targeting of drug sites. These peptides play a crucial role in the innate immunity of insects, offering protection against bacteria, fungi, and parasites. Knottins have garnered considerable interest as promising contenders for drug development due to their ability to bridge the gap between small molecules and protein-based biopharmaceuticals, effectively addressing the therapeutic limitations of both modalities. This work presents the isolation and identification of a novel antimicrobial peptide derived from Monochamus alternatus. The cDNA encodes a 56-amino acid knottin propeptide, while the mature peptide comprises only 34 amino acids. We have labeled this knottin peptide as MaK. Using chemically synthesized MaK, we evaluated its hemolytic activity, thermal stability, antibacterial properties, and efficacy against nematodes. The results of this study indicate that MaK is an exceptionally effective knottin-type peptide. It demonstrates low toxicity, superior stability, potent antibacterial activity, and the ability to suppress pine wood nematodes. Consequently, these findings suggest that MaK has potential use in developing innovative therapeutic agents to prevent and manage pine wilt disease.

Identification of a common epitope in knottins and phospholipases D present in Loxosceles sp venom by a monoclonal antibody.

In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.

Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1ß Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer.

Phα1ß (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1ß administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1ß and its 15N-labeled analogue. Spatial structure and dynamics of Phα1ß were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1ß structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1ß has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1ß significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1ß as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Cystine Knot Peptides with Tuneable Activity and Mechanism.

Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function.

Pilot-phase PET/CT study targeting integrin αvß6 in pancreatic cancer patients using the cystine-knot peptide-based 18F-FP-R01-MG-F2.

PURPOSE: A novel cystine-knot peptide-based PET radiopharmaceutical, 18F-FP-R01-MG-F2 (knottin), was developed to selectively bind to human integrin αvß6 which is overexpressed in pancreatic cancer. The purpose of this study is to evaluate the safety, biodistribution, dosimetry, and lesion uptake of 18F-FP-R01-MG-F2 in patients with pancreatic cancer. METHODS: Fifteen patients (6 men, 9 women) with histologically confirmed pancreatic cancer were prospectively enrolled and underwent knottin PET/CT between March 2017 and February 2021 (ClinicalTrials.gov Identifier NCT02683824). Vital signs and laboratory results were collected before and after the imaging scans. Maximum standardized uptake values (SUVmax) and mean SUV (SUVmean) were measured in 24 normal tissues and pancreatic cancer lesions for each patient. From the biodistribution data, the organ doses and whole-body effective dose were calculated using OLINDA/EXM software. RESULTS: There were no significant changes in vital signs or laboratory values that qualified as adverse events or serious adverse events. At 1 h post-injection, areas of high 18F-FP-R01-MG-F2 uptake included the pituitary gland, stomach, duodenum, kidneys, and bladder (average SUVmean: 9.7-14.5). Intermediate uptake was found in the normal pancreas (average SUVmean: 4.5). Mild uptake was found in the lungs and liver (average SUVmean < 1.0). The effective dose was calculated to be 2.538 × 10-2 mSv/MBq. Knottin PET/CT detected all known pancreatic tumors in the 15 patients, although it did not detect small peri-pancreatic lymph nodes of less than 1 cm in short diameter in two of three patients who had lymph node metastases at surgery. Knottin PET/CT detected distant metastases in the lungs (n = 5), liver (n = 4), and peritoneum (n = 2), confirmed by biopsy and/or contrast-enhanced CT. CONCLUSION: 18F-FP-R01-MG-F2 is a safe PET radiopharmaceutical with an effective dose comparable to other diagnostic agents. Evaluation of the primary pancreatic cancer and distant metastases with 18F-FP-R01-MG-F2 PET is feasible, but larger studies are required to define the role of this approach. TRIAL REGISTRATION: NCT02683824.

Arthropod toxins inhibiting Ca2+ and Na+ channels prevent AC-1001 H3 peptide-induced apoptosis.

Peptide toxins of arthropods are one of the potential sources of bioactive substances. Toxins are able to bind to calcium channels and block them. Ca2+ ions play an important role in many cell processes, in particular, in apoptosis. In this work, we study the effect of some arthropod toxins on intracellular processes associated with the induction of apoptosis. Synthetic analogs of U5 -scytotoxin-Sth1a, ω-hexatoxin-Hv1a, ω-theraphotoxin-Hhn2a, and µ-agatoxin-Aa1a toxins-inhibitors of calcium L, P, and Q channels and sodium channels were used in the study. Apoptosis was induced by AC-1001 H3 peptide. We study the effect of toxins on the level of apoptosis, ROS, mitochondrial potential, GSH, and ATP in CHO-K1 cells. We show that all the tested toxins are able to dose dependently block the induction of apoptosis triggered by AC-1001 H3 and reduce the level of natural apoptosis in CHO-K1 cells. Cell incubation with apoptosis inducer AC-1001 H3 in the presence and absence of toxins causes an increase in the intracellular concentrations of ROS, ATP, and mitochondrial potential and decreases the GSH concentration. The present study reveals the antiapoptotic effect of a number of arthropod peptide toxins. The toxins studied can represent a novel approach used in the treatment of pathologies associated with the activation of apoptotic mechanisms.

Targeting the tumor vasculature with engineered cystine-knot miniproteins.

The extra domain B splice variant (EDB) of human fibronectin selectively expressed in the tumor vasculature is an attractive target for cancer imaging and therapy. Here, we describe the generation and characterization of EDB-specific optical imaging probes. By screening combinatorial cystine-knot miniprotein libraries with phage display technology we discover exquisitely EDB-specific ligands that share a distinctive motif. Probes with a binding constant in the picomolar range are generated by chemical oligomerization of selected ligands and fluorophore conjugation. We show by fluorescence imaging that the probes stain EDB in tissue sections derived from human U-87 MG glioblastoma xenografts in mice. Moreover, we demonstrate selective accumulation and retention of intravenously administered probes in the tumor tissue of mice with U-87 MG glioblastoma xenografts by in vivo and ex vivo fluorescence imaging. These data warrants further pursuit of the selected cystine-knot miniproteins for in vivo imaging applications.

Weaponisation 'on the fly': Convergent recruitment of knottin and defensin peptide scaffolds into the venom of predatory assassin flies.

Many arthropod venom peptides have potential as bioinsecticides, drug leads, and pharmacological tools due to their specific neuromodulatory functions. Assassin flies (Asilidae) are a family of predaceous dipterans that produce a unique and complex peptide-rich venom for killing insect prey and deterring predators. However, very little is known about the structure and function of their venom peptides. We therefore used an E. coli periplasmic expression system to express four disulfide-rich peptides that we previously reported to exist in venom of the giant assassin fly Dolopus genitalis. After purification, each recombinant peptide eluted from a C18 column at a position closely matching its natural counterpart, strongly suggesting adoption of the native tertiary fold. Injection of purified recombinant peptides into blowflies (Lucilia cuprina) and crickets (Acheta domestica) revealed that two of the four recombinant peptides, named rDg3b and rDg12, inhibited escape behaviour in a manner that was rapid in onset (<1 min) and reversible. Homonuclear NMR solution structures revealed that rDg3b and rDg12 adopt cystine-stabilised α/ß defensin and inhibitor cystine knot folds, respectively. Although the closest known homologues of rDg3b at the level of primary structure are dipteran antimicrobial peptides such as sapecin and lucifensin, a DALI search showed that the tertiary structure of rDg3b most closely resembles the KV11.1-specific α-potassium channel toxin CnErg1 from venom of the scorpion Centruroides noxius. This is mainly due to the deletion of a large, unstructured loop between the first and second cysteine residues present in Dg3b homologues from non-asiloid, but not existing in asiloid, species. Patch-clamp electrophysiology experiments revealed that rDg3b shifts the voltage-dependence of KV11.1 channel activation to more depolarised potentials, but has no effect on KV1.3, KV2.1, KV10.1, KCa1.1, or the Drosophila Shaker channel. Although rDg12 shares the inhibitor cystine knot structure of many gating modifier toxins, rDg12 did not affect any of these KV channel subtypes. Our results demonstrate that multiple disulfide-rich peptide scaffolds have been convergently recruited into asilid and other animal venoms, and they provide insight into the molecular evolution accompanying their weaponisation.

In Silico Investigation of the Binding of MCoTI-II Plant Defense Knottin to the γ-NGF Serine Protease of the 7S Nerve Growth Factor Complex and Biological Activity of Its NGF Mimetic Properties.

Nerve growth factor (NGF) is an endogenously produced polypeptide that promotes the differentiation, survival, and repair of neurons in the central and peripheral nervous systems. While trophic proteins hold promise for the treatment of neuronal injury and disease, use of NGF is limited by its large molecular weight, lack of permeability through the blood-brain barrier, and peripheral side effects. Previously, we found that an extract of the Momordica cochinchinensis seed stimulated PC-12 neurite outgrowth. Bioactivity-guided fractioning of the seed extract suggested that the NGF mimetic agent was one of few defined proteins from this plant: one group being the defense Knottins and the other group of the lowest mass is the potent trypsin inhibitor MCoTI-II. Here, the NGF mimetic potential of this latter protein was investigated using two concurrent but different approaches. A biological study used recombinant purified MCoTI-II, which when tested in rat PC-12 cells grown on collagen, failed to initiate outgrowth relative to the positive control 7S NGF. In a separate computational study, the possibility was investigated such that MCoTI-II could exert an effect through binding to the serine protease γ-NGF subunit of the 7S NGF complex, analogous to its binding to its native receptor trypsin. Molecular dynamics simulations showed that MCoTI-II can bind stably to γ-NGF for >350 ns. Modeling indicated that this interaction could sterically inhibit 7S NGF complex formation, potentially altering the equilibrium between inactive complexed and free active NFG protein. In conclusion, the biological study now excludes the MCoTI-II protein as the NGF mimetic factor in the Momordica extract, an important and required step to identify the active component in this seed. On the other hand, the theoretical study has revealed a novel observation that may be of use in the development of strategies to affect NGF activity.

Fluorescence Imaging of Peripheral Nerves by a Nav1.7-Targeted Inhibitor Cystine Knot Peptide.

Twenty million Americans suffer from peripheral nerve injury caused by trauma and medical disorders, resulting in a broad spectrum of potentially debilitating side effects. In one out of four cases, patients identify surgery as the root cause of their nerve injury. Particularly during tumor resections or after traumatic injuries, tissue distortion and poor visibility can challenge a surgeon's ability to precisely locate and preserve peripheral nerves. Intuitively, surgical outcomes would improve tremendously if nerves could be highlighted using an exogeneous contrast agent. In clinical practice, however, the current standard of care-visual examination and palpation-remains unchanged. To address this unmet clinical need, we explored the expression of voltage-gated sodium channel Nav1.7 as an intraoperative marker for the peripheral nervous system. We show that expression of Nav1.7 is high in peripheral nerves harvested from both human and mouse tissue. We further show that modification of a Nav1.7-selective peptide, Hsp1a, can serve as a targeted vector for delivering a fluorescent sensor to the peripheral nervous system. Ex vivo, we observe a high signal-to-noise ratio for fluorescently labeled Hsp1a in both histologically prepared and fresh tissue. Using a surgical fluorescent microscope, we show in a simulated clinical scenario that the identification of mouse sciatic nerves is possible, suggesting that fluorescently labeled Hsp1a tracers could be used to discriminate nerves from their surrounding tissues in a routine clinical setting.

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVß3 and α5ß1.

Targeting both integrins αVß3 and α5ß1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.5F binds selectively to αVß3 and α5ß1, whereas knottin 2.5D is αVß3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with αVß3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages αVß3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.

A knottin scaffold directs the CXC-chemokine-binding specificity of tick evasins.

Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or "one-to-many" mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine-EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine-binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR+ CXC-chemokines, whereas the second class bound both ELR+ and ELR- CXC-chemokines, in several cases including CXC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel ß-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine-binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine-binding tick EVAs achieve class specificity but also engage in promiscuous binding.

Tying pest insects in knots: the deployment of spider-venom-derived knottins as bioinsecticides.

Spider venoms are complex chemical arsenals that contain a rich variety of insecticidal toxins. However, the major toxin class in many spider venoms is disulfide-rich peptides known as knottins. The knotted three-dimensional fold of these mini-proteins provides them with exceptional chemical and thermal stability as well as resistance to proteases. In contrast with other bioinsecticides, which are often slow-acting, spider knottins are fast-acting neurotoxins. In addition to being potently insecticidal, some knottins have exceptional taxonomic selectivity, being lethal to key agricultural pests but innocuous to vertebrates and beneficial insects such as bees. The intrinsic oral activity of these peptides, combined with the ability of aerosolized knottins to penetrate insect spiracles, has enabled them to be developed commercially as eco-friendly bioinsecticides. Moreover, it has been demonstrated that spider-knottin transgenes can be used to engineer faster-acting entomopathogens and insect-resistant crops. © 2019 Society of Chemical Industry.

Detecting Vulnerable Atherosclerotic Plaques by 68Ga-Labeled Divalent Cystine Knot Peptide.

Integrin αvß3 has been considered as a promising biomarker for vulnerable atherosclerotic plaques, and it is highly expressed by those instability-associated factors, such as macrophages, vessel endothelial cells, and smooth muscle cells. Our previous study successfully showed that the 64Cu-labeled divalent (containing two RGD motifs) cystine knot peptide, 64Cu-NOTA-3-4A, had high binding affinity and specificity in targeting vulnerable carotid atherosclerotic plaques with increased αvß3 levels. Therefore, considering that 68Ga has excellent nuclear physical properties for positron emission tomography (PET), this study aimed to investigate the feasibility of using 68Ga-NOTA-3-4A for PET study of vulnerable atherosclerotic plaques. The vulnerable carotid atherosclerotic plaques were induced and maintained in ApoE-/- mice through carotid artery ligation and a high-fat diet. Divalent knottin peptide 3-4A was synthesized through solid-phase peptide synthesis chemistry and radiolabeled with 68Ga after being conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The probe stability was analyzed in phosphate buffered saline (PBS) buffer and mouse serum. ApoE-/- mice with atherosclerotic plaques ( n = 4) were imaged by PET/CT at 1 and 2 h after the tail vein injection of 68Ga-NOTA-3-4A. The targeting specificity was determined by coinjection of 68Ga-NOTA-3-4A and nonradioactive c(RGDyK) peptide. The carotid artery tissues were removed, and immunofluorescent staining was performed to evaluate αvß3 integrin expression. It was found that 68Ga-NOTA-3-4A displayed high stability in both PBS buffer and mouse serum. Small animal PET/CT images and quantification analysis indicated the quick and high plaque uptake of 68Ga-NOTA-3-4A (6.67 ± 1.44 and 2.97 ± 0.46%ID/g at 1 and 2 h, respectively). The plaque-to-normal artery ratio was 15.88 and 9.90 at 1 and 2 h, respectively. Furthermore, the plaque accumulation of 68Ga-NOTA-3-4A was significantly inhibited via coinjection of c(RGDyK). Finally, immunostaining identified integrin αvß3 expressed by macrophages, vessel endothelial cells, and smooth muscle cells. In summary, 68Ga-NOTA-3-4A has high potential to be a promising PET tracer for imaging vulnerable atherosclerotic plaques.

Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel NaV1.7.

Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate NaV1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.

Protecting Encapsulin Nanoparticles with Cysteine-Knot Miniproteins.

A big hurdle for the use of protein-based drugs is that they are easily degraded by proteases in the human body. In an attempt to solve this problem, we show the possibility to functionalize TM encapsulin nanoparticles with an mEETI-II knottin miniprotein from the cysteine-stabilized knot class. The resulting particles did not show aggregation and retained part of their protease inhibitive function. This imposes a protection toward protease, in this case, trypsin, degradation of the protein cage. The used chemistry is easy to apply and thus suitable to protect other protein systems from degradation. In addition, this proof of principle opens up the use of other knottins or cysteine-stabilized knots, which can be attached to protein cages to create a heterofunctionalized protein nanocage. This allows specific targeting and tumor suppression among other types of functionalization. Overall, this is a promising strategy to protect a protein of interest which brings oral administration of protein-based drugs one step closer.

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.

Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.

Development and Preclinical Validation of a Cysteine Knottin Peptide Targeting Integrin αvß6 for Near-infrared Fluorescent-guided Surgery in Pancreatic Cancer.

Purpose: Intraoperative near-infrared fluorescence (NIRF) imaging could help stratification for the proper primary treatment for patients with pancreatic ductal adenocarcinoma (PDAC), and achieve complete resection, as it allows visualization of cancer in real time. Integrin αvß6, a target specific for PDAC, is present in >90% of patients, and is able to differentiate between pancreatitis and PDAC. A clinically translatable αvß6-targeting NIRF agent was developed, based on a previously developed cysteine knottin peptide for PET imaging, R01-MG, and validated in preclinical mouse models.Experimental Design: The applicability of the agent was tested for cell and tissue binding characteristics using cell-based plate assays, subcutaneous, and orthotopic pancreatic models, and a transgenic mouse model of PDAC development (Pdx1-Cretg/+;KRasLSL G12D/+;Ink4a/Arf-/-). IRDye800CW was conjugated to R01-MG in a 1:1 ratio. R01-MG-IRDye800, was compared with a control peptide and IRDye800 alone.Results: In subcutaneous tumor models, a significantly higher tumor-to-background ratio (TBR) was seen in BxPC-3 tumors (2.5 ± 0.1) compared with MiaPaCa-2 (1.2 ± 0.1; P < 0.001), and to the control peptide (1.6 ± 0.4; P < 0.005). In an orthotopic tumor model, tumor-specific uptake of R01-MG-IRDye800 was shown compared with IRDye800 alone (TBR 2.7 vs. 0.86). The fluorescent signal in tumors of transgenic mice was significantly higher, TBR of 3.6 ± 0.94, compared with the normal pancreas of wild-type controls, TBR of 1.0 ± 0.17 (P < 0.001).Conclusions: R01-MG-IRDye800 shows specific targeting to αvß6, and holds promise as a diagnostic and therapeutic tool to recognize PDAC for fluorescence-guided surgery. This agent can help improve the stratification of patients for a potentially curative, margin-negative resection. Clin Cancer Res; 24(7); 1667-76. ©2018 AACR.

KNOTTIN: the database of inhibitor cystine knot scaffold after 10 years, toward a systematic structure modeling.

Knottins, or inhibitor cystine knots (ICKs), are ultra-stable miniproteins with multiple applications in drug design and medical imaging. These widespread and functionally diverse proteins are characterized by the presence of three interwoven disulfide bridges in their structure, which form a unique pseudoknot. Since 2004, the KNOTTIN database (www.dsimb.inserm.fr/KNOTTIN/) has been gathering standardized information about knottin sequences, structures, functions and evolution. The website also provides access to bibliographic data and to computational tools that have been specifically developed for ICKs. Here, we present a major upgrade of our database, both in terms of data content and user interface. In addition to the new features, this article describes how KNOTTIN has seen its size multiplied over the past ten years (since its last publication), notably with the recent inclusion of predicted ICKs structures. Finally, we report how our web resource has proved usefulness for the researchers working on ICKs, and how the new version of the KNOTTIN website will continue to serve this active community.

High-Affinity RGD-Knottin Peptide as a New Tool for Rapid Evaluation of the Binding Strength of Unlabeled RGD-Peptides to αvß3, αvß5, and α5ß1 Integrin Receptors.

We describe a highly sensitive competition ELISA to measure integrin-binding of RGD-peptides in high-throughput without using cells, ECM-proteins, or antibodies. The assay measures (nonlabeled) RGD-peptides' ability to inhibit binding of a biotinylated "knottin"-RGD peptide to surface-immobilized integrins and, thus, enables quantification of the binding strength of high-, medium-, and low-affinity RGD-binders. We introduced the biotinylated knottin-RGD peptide instead of biotinylated cyclo[RGDfK] (as reported by Piras et al.), as integrin-binding was much stronger and clearly detectable for all three integrins. In order to maximize sensitivity and cost-efficiency, we first optimized several parameters, such as integrin-immobilization levels, knottin-RGD concentration, buffer compositions, type of detection tag (biotin, His- or cMyc-tag), and spacer length. We thereby identified two key factors, that is, (i) the critical spacer length (longer than Gly) and (ii) the presence of Ca2+ and Mg2+ in all incubation and washing buffers. Binding of knottin-RGD peptide was strongest for αvß3 but also detectable for both αvß5 and α5ß1, while binding of biotinylated cyclo[RGDfK] was very weak and only detectable for αvß3. For assay validation, we finally determined IC50 values for three unlabeled peptides, that is: (i) linear GRGDS, (ii) cyclo[RGDfK], and (iii) the knottin-RGD itself for binding to three different integrin receptors (αvß3, αvß5, α5ß1). Major benefits of the novel assay are (i) the extremely low consumption of integrin (50 ng/peptide), (ii) the fact that neither antibodies/ECM-proteins nor integrin-expressing cells are required for detection, and (iii) its suitability for high-throughput screening of (RGD-)peptide libraries.