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.

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.

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.

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.

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.

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.

[Prospects for the design of new therapeutically significant protease inhibitors based on knottins and sunflower seed trypsin inhibitor (SFTI 1)]. / Perspektivy sozdaniia novykh ingibitorov terapevticheski znachimykh serinovykh proteaz na osnove knottinov i peptidnogo ingibitora tripsina iz semian podsolnechnika (SFTI 1).

Plant seed knottins, mainly from the Cucurbitacea family, and sunflower seed trypsin inhibitor (SFTI 1) are the most low-molecular canonical peptide inhibitors of serine proteases. High efficiency of inhibition of various serine proteases, structure rigidity together with the possibility of limited variations of amino acid sequences, high chemical stability, lack of toxic properties, opportunity of production by either chemical synthesis or use of heterologous expression systems make these inhibitors attractive templates for design of new compounds for regulation of therapeutically significant serine protease activities. Hence the design of such compounds represents a prospective research field. The review considers structural characteristics of these inhibitors, their properties, methods of preparation and design of new analogs. Examples of successful employment of natural serine protease inhibitors belonging to knottin family and SFTI 1 as templates for the design of highly specific inhibitors of certain proteases are given.

Integrin-Targeting Knottin Peptide-Drug Conjugates Are Potent Inhibitors of Tumor Cell Proliferation.

Antibody-drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide-drug delivery, which has the potential for increased tumor penetration and facile synthesis. We report a knottin peptide-drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor-associated integrins. This KDC binds to tumor cells with low-nanomolar affinity, is internalized by an integrin-mediated process, releases its payload intracellularly, and is a highly potent inhibitor of brain, breast, ovarian, and pancreatic cancer cell lines. Notably, these features enable this KDC to bypass a gemcitabine-resistance mechanism found in pancreatic cancer cells. This work expands the therapeutic relevance of knottin peptides to include targeted drug delivery, and further motivates efforts to expand the drug-conjugate toolkit to include non-antibody protein scaffolds.

Cystine-knot peptides: emerging tools for cancer imaging and therapy.

Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature's repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.

Combinatorial optimization of cystine-knot peptides towards high-affinity inhibitors of human matriptase-1.

Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor) and SOTI (Spinacia oleracea trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.

Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma.

Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to αvß3, αvß5, and α5ß1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α5ß1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging.

The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels.

One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large ß hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel.

Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis.

The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization.

Preliminary evaluation of (177)Lu-labeled knottin peptides for integrin receptor-targeted radionuclide therapy.

PURPOSE: Cystine knot peptides (knottins) 2.5D and 2.5F were recently engineered to bind integrin receptors with high affinity and specificity. These receptors are overexpressed on the surface of a variety of malignant human tumor cells and tumor neovasculature. In this study, 2.5D and 2.5F were labeled with a therapeutic radionuclide, (177)Lu, and the resulting radiopeptides were then evaluated as potential radiotherapeutic agents in a murine model of human glioma xenografts. METHODS: Knottins 2.5D and 2.5F were synthesized using solid phase peptide synthesis, folded in vitro, and site-specifically coupled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) at their N terminus for (177)Lu radiolabeling. The stability of the radiopeptides (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F was tested in both phosphate-buffered saline (PBS) and mouse serum. Cell uptake assays of the radiolabeled peptides were performed in U87MG integrin-expressing human glioma cells. The biodistribution studies of both (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F were examined in U87MG tumor-bearing athymic nu/nu mice. Radiation absorbed doses for the major tissues of a human adult male were calculated based on the mouse biodistribution results. RESULTS: DOTA-2.5D and DOTA-2.5F were labeled with (177)Lu at over 55% efficiency. High radiochemical purity for both radiocomplexes (> 95%) could be achieved after high performance liquid chromatography (HPLC) purification. Both radiopeptides were stable in PBS and mouse serum. Compared to (177)Lu-DOTA-2.5D (0.39 and 0.26 %ID/g at 2 and 24 h, respectively), (177)Lu-DOTA-2.5F showed much higher tumor uptake (2.16 and 0.78 %ID/g at 2 and 24 h, respectively). It also displayed higher tumor to blood ratios than that of (177)Lu-DOTA-2.5D (31.8 vs 18.7 at 24 h and 52.6 vs 20.6 at 72 h). Calculation of radiodosimetry for (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F suggested that tumor and kidney were tissues with the highest radiation absorbed doses. Moreover, (177)Lu-DOTA-2.5F had a higher tumor to kidney radiation absorbed dose ratio than that of (177)Lu-DOTA-2.5D. CONCLUSION: Cystine knot peptides can be successfully radiolabeled with (177)Lu for potential therapeutic applications. Knottin 2.5F labeled with (177)Lu exhibits favorable distribution in murine U87MG xenograft model; thus, it is a promising agent for radionuclide therapy of integrin-positive tumors.