Light Chain Protease Research Articles

Effect of electroacupuncture on expression of autophagy-related proteins in nucleus pulposus of cervical spondylosis rabbits.

To observe the effects of electroacupuncture (EA) on the morphological changes of intervertebral disc tissues, apoptosis of nucleus pulposus cells, and the protein expression of Unc-51 like autophagy-activated kinase 1 (ULK1), homologous series of yeast Atg6 (Beclin1), and light chain protease complication 3 type (LC3) in nucleus pulposus tissue of cervical spondylosis rabbits, so as to explore the role of cellular autophagy in EA treatment of cervical spondylosis. A total of 24 New Zealand white rabbits were randomly divided into blank, model and EA groups, with 8 rabbits in each group. In the EA group, both sides of the cervical (C)3-C6 "Jiaji" (EX-B2) were stimulated by EA (2 Hz/100 Hz, 1 mA) for 25 min, once daily for 5 days in a course, with a 2-day interval between courses, totaling 4 treatment courses. X-ray was used to assess cervical spine radiographic changes and evaluate radiographic scores；transmission electron microscopy was used to observe ultrastructural changes in nucleus pulposus cells；HE staining was used to observe morphological changes of intervertebral disc tissues and conduct pathological scoring；TUNEL staining was used to observe apoptosis rate of nucleus pulposus cells；Western blot was performed to detect protein expression levels of ULK1, Beclin1, and LC3 in nucleus pulposus tissue. Compared with the blank group, rabbits in the model group showed significantly higher cervical spine radiographic scores (P<0.01), higher pathological scores of intervertebral disc tissues (P<0.05), increased apoptosis rate of nucleus pulposus cells (P<0.01), and decreased expression levels of ULK1, Beclin1, and LC3Ⅱ proteins in nucleus pulposus tissue (P<0.05). Compared with the model group, the EA group showed significantly lower pathological scores of intervertebral discs (P<0.05), lower apoptosis rate of nucleus pulposus cells (P<0.01), and higher protein expression levels of ULK1, Beclin1, and LC3Ⅱ in nucleus pulposus tissue (P<0.01). Rabbits in the blank control group exhibited generally normal organelle structures in nucleus pulposus tissues with few autophagic vacuoles, indicative of early stages of autophagy；while those in the model group showed disrupted organelle structures with cytoplasmic condensation and those in the EA group exhibited autophagosomes with double-membrane structures in nucleus pulposus tissues. EA promotes the expression of ULK1, Beclin1, and LC3Ⅱ proteins in nucleus pulposus tissues, reduces apoptosis of nucleus pulposus cells, and improves intervertebral disc degeneration.

Guasha improves knee osteoarthritis by inhibiting chondrocyte apoptosis and regulating expression of autophagy-related genes and proteins in rats.

To observe the effect of Guasha on inflammation factors, apoptosis and autophagy in the cartilage tissue of knee joint in rats with knee osteoarthritis (KOA), so as to explore its mechanisms underlying improvement of KOA. A total of 51 male SD rats were randomized into three groups：blank control, KOA model and Guasha (n= 17 in each group) . The rats in the blank control group received intra-articular injection of 0.9% NaCl solution in the right knee joint. The KOA model was established by intraarticular injection of glutamate sodium iodoacetic acid in the right knee joint. For rats of the Guasha group, Guasha (at a frequency of 1 time/s, and an applied pressure of 0.3-0.5 kgf) was applied to "Yanglingquan" (GB34) and "Xuehai"(SP10) areas of the right leg, once every other day, for 7 consecutive sessions. The circumference of the right knee was measured, The histopathological changes of right knee cartilage were observed after H.E. staining. The contents of inflammatory factors interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the right knee articular cartilage tissue were assayed using ELISA. The expression levels of autophagy-related key molecule Beclin-1 (homologous series of yeast Atg6), light chain protease complication 3 type II/I (LC3II/LC3 I), ubiquitin binding factor 62 (P62) and cysteine aspartate protease-3 (Caspase-3) mRNAs and proteins of the right knee articular cartilage tissue were measured using real-time fluorescent quantitative PCR and Western blot, separately. The apoptosis of chondrocytes was assayed using TUNEL staining, and the immunoactivity of LC3 determined using immunofluorescence staining. After modeling, the right knee circumfe-rence of the model and Guasha groups was significantly increased compared with the blank control group (P<0.01), and after the intervention, the knee circumference of the Guasha group was markedly decreased in comparison with that of the model group (P<0.05). Results of H.E. staining showed obvious degeneration and defects in the cartilage tissue, necrosis of a large number of chondrocytes, fibrous hyperplasia, accompanied by inflammatory cell infiltration, osteoclast increase, fibroplasia and bone trabecular destruction in the model group, which was relatively milder in the Guasha group. Compared with the blank control group, the expression of Beclin-1 and LC3 mRNAs and proteins, and LC immunofluorescence intensity in the right knee articular cartilage tissue were significantly down-regulated (P<0.01, P<0.001), whereas the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, contents of IL-1β and TNF-α in the right knee articular cartilage tissue considerably increased (P<0.01, P<0.001) in the model group. In contrast to the model group, the Guasha group had an apparent increase in the expression levels of Beclin-1 and LC3 mRNAs and proteins and LC immunofluorescence intensity in the right knee articular cartilage tissue (P<0.05), and a pronounced decrease in the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, and contents of IL-1β and TNF-α in the right knee articular cartilage tissue (P<0.05, P<0.01). Guasha stimulation of GB34 and SP10 can improve joint cartilage damage in KOA rats, which may be associated with its functions in inhibiting the excessive release of inflammatory factors and apoptosis, possibly by down-regulating the expression of P62 and Caspase-3 mRNAs and proteins and up-regulating the expression of Beclin-1 and LC3 mRNAs and proteins, and by promoting autophagy of chondrocytes.

Selective Expression of a SNARE-Cleaving Protease in Peripheral Sensory Neurons Attenuates Pain-Related Gene Transcription and Neuropeptide Release.

Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1; CALCB, calcitonin gene-related peptide 2; HTR3A, 5-hydroxytryptamine receptor 3A; NPY2R, neuropeptide Y receptor Y2; GPR52, G protein-coupled receptor 52; SCN9A, sodium voltage-gated channel alpha subunit 9; TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief.

Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function.

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA.

The extreme toxicity of botulinum neurotoxins (BoNTs) relies on their specific cleavage of SNARE proteins, which eventually leads to muscle paralysis. One newly identified mosaic toxin, BoNT/HA (aka H or FA), cleaves VAMP-2 at a unique position between residues L54 and E55, but the molecular basis underlying VAMP-2 recognition of BoNT/HA remains poorly characterized. Here, we report a ∼2.09 Å resolution crystal structure of the light chain protease domain of BoNT/HA (LC/HA). Structural comparison between LC/HA and LC of BoNT/F1 (LC/F1) reveals distinctive hydrophobic and electrostatic features near the active sites, which may explain their different VAMP-2 cleavage sites. When compared to BoNT/F5 that cleaves VAMP-2 at the same site as BoNT/HA, LC/HA displays higher affinity for VAMP-2, which could be caused by their different surface charge properties surrounding a VAMP-2 exosite-binding cleft. Furthermore, systematic mutagenesis studies on VAMP-2 and structural modeling demonstrate that residues R47 to K59 spanning the cleavage site in VAMP-2 may adopt a novel extended conformation when interacting with LC/HA and LC/F5. Taken together, our structure provides new insights into substrate recognition of BoNT/HA and paves the way for rational design of small molecule or peptide inhibitors against LC/HA.

Deubiquitinating enzyme VCIP135 dictates the duration of botulinum neurotoxin type A intoxication

Botulism is characterized by flaccid paralysis, which can be caused by intoxication with any of the seven known serotypes of botulinum neurotoxin (BoNT), all of which disrupt synaptic transmission by endoproteolytic cleavage of SNARE proteins. BoNT serotype A (BoNT/A) has the most prolonged or persistent effects, which can last several months, and exerts its effects by specifically cleaving and inactivating SNAP25. A major factor contributing to the persistence of intoxication is the long half-life of the catalytic light chain, which remains enzymatically active months after entry into cells. Here we report that BoNT/A catalytic light chain binds to, and is a substrate for, the ubiquitin ligase HECTD2. However, the light chain evades proteasomal degradation by the dominant effect of a deubiquitinating enzyme, VCIP135/VCPIP1. This deubiquitinating enzyme binds BoNT/A light chain directly, with the two associating in cells through the C-terminal 77 amino acids of the light chain protease. The development of specific DUB inhibitors, together with inhibitors of BoNT/A proteolytic activity, may be useful for reducing the morbidity and public health costs associated with BoNT/A intoxication and could have potential biodefense implications.

Metal Ions Effectively Ablate the Action of Botulinum Neurotoxin A.

Botulinum neurotoxin serotype A (BoNT/A) causes a debilitating and potentially fatal illness known as botulism. The toxin is also a bioterrorism threat, yet no pharmacological antagonist to counteract its effects has reached clinical approval. Existing strategies to negate BoNT/A intoxication have looked to antibodies, peptides, or organic small molecules as potential therapeutics. In this work, a departure from the traditional drug discovery mindset was pursued, in which the enzyme's susceptibility to metal ions was exploited. A screen of a series of metal salts showed marked inhibitory activity of group 11 and 12 metals against the BoNT/A light chain (LC) protease. Enzyme kinetics revealed that copper (I) and (II) cations displayed noncompetitive inhibition of the LC (Ki ≈ 1 μM), while mercury (II) cations were 10-fold more potent. Crystallographic and mutagenesis studies elucidated a key binding interaction between Cys165 on BoNT/A LC and the inhibitory metals. As potential copper prodrugs, ligand-copper complexes were examined in a cell-based model and were found to prevent BoNT/A cleavage of the endogenous protein substrate, SNAP-25, even at low μM concentrations of complexes. Further investigation of the complexes suggested a bioreductive mechanism causing intracellular release of copper, which directly inhibited the BoNT/A protease. In vivo experiments demonstrated that copper (II) dithiocarbamate and bis(thiosemicarbazone) complexes could delay BoNT/A-mediated lethality in a rodent model, indicating their potential for treating the harmful effects of BoNT/A intoxication. Our studies illustrate that metals can be therapeutically viable enzyme inhibitors; moreover, enzymes that share homology with BoNT LCs may be similarly targeted with metals.

Cellular Protection of SNAP-25 against Botulinum Neurotoxin/A: Inhibition of Thioredoxin Reductase through a Suicide Substrate Mechanism.

Botulium neurotoxins (BoNTs) are among the most lethal toxins known to man. They are comprised of seven serotypes with BoNT/A being the most deadly; yet, there is no approved therapeutic for their intoxication or one that has even advanced to clinical trials. Botulinum neurotoxicity is ultimately governed through light chain (LC) protease SNARE protein cleavage leading to a loss of neurotransmitter release. Pharmacological attempts to ablate BoNT/A intoxication have sought to either nullify cellular toxin entry or critical biochemical junctions found within its intricate mechanism of action. In these regards, reports have surfaced of nonpeptidic small molecule inhibitors, but few have demonstrated efficacy in neutralizing cellular toxicity, a key prerequisite before rodent lethality studies can be initiated. On the basis of a lead discovered in our BoNT/A cellular assay campaign, we investigated a family of N-hydroxysuccinimide inhibitors grounded upon structure activity relationship (SAR) fundamentals. Molecules stemming from this SAR exercise were theorized to be protease inhibitors. However, this proposition was overturned on the basis of extensive kinetic analysis. Unexpectedly, inhibitor data pointed to thioredoxin reductase (TrxR), an essential component required for BoNT protease translocation. Also unforeseen was the inhibitors' mechanism of action against TrxR, which was found to be brokered through a suicide-mechanism utilizing quinone methide as the inactivating element. This new series of TrxR inhibitors provides an alternative means to negate the etiological agent responsible for BoNT intoxication, the LC protease.

A reduced graphene oxide-Au based electrochemical biosensor for ultrasensitive detection of enzymatic activity of botulinum neurotoxin A

Botulinum neurotoxin serotype A (BoNT/A) is a type of neurotoxin which is able to cause fatal paralytic illness botulism in a low dosage. Therefore, there is a great need to develop an ultrasensitive bioassay to detect its active state for early diagnostics and prevention. This paper presents a reduced graphene oxide (rGO)/Au electrode based electrochemical biosensor for ultrasensitive detection of BoNT serotype A light chain (BoNT-LcA) protease activity. The fabricated rGO/Au electrode provides a robust and biocompatible platform with enhanced electron transfer capability and large area for peptide immobilization. SNAP-25-GFP peptide substrate is firstly immobilized on rGO surface via pyrenebutyric acid (PA) linker. The addition of BoNT-LcA could specifically cut SNAP-25-GFP at the cleavage sites to release the cut section from the electrode surface. This enzymatic activity of BoNT-LcA on SNAP-25-GFP peptide substrate could be detected by monitoring the enhanced redox probe transfer rate by differential pulse voltammetry (DPV) with a linear detection range from 1pg/mL to 1ng/mL and the limit of detection (LOD) for BoNT-LcA is around 8.6pg/mL. The specificity of this biosensor is demonstrated with BoNT-LcB and heat-treated BoNT-LcA. Moreover, the experiments for BoNT-LcA detection in milk samples demonstrate the feasibility of this biosensor in complex matrix.

Coagulation factor XII protease domain crystal structure

BackgroundCoagulation factor XII is a serine protease that is important for kinin generation and blood coagulation, cleaving the substrates plasma kallikrein and FXI.ObjectiveTo investigate FXII zymogen activation and substrate recognition by determining the crystal structure of the FXII protease domain.Methods and resultsA series of recombinant FXII protease constructs were characterized by measurement of cleavage of chromogenic peptide and plasma kallikrein protein substrates. This revealed that the FXII protease construct spanning the light chain has unexpectedly weak proteolytic activity compared to β-FXIIa, which has an additional nine amino acid remnant of the heavy chain present. Consistent with these data, the crystal structure of the light chain protease reveals a zymogen conformation for active site residues Gly193 and Ser195, where the oxyanion hole is absent. The Asp194 side chain salt bridge to Arg73 constitutes an atypical conformation of the 70-loop. In one crystal form, the S1 pocket loops are partially flexible, which is typical of a zymogen. In a second crystal form of the deglycosylated light chain, the S1 pocket loops are ordered, and a short α-helix in the 180-loop of the structure results in an enlarged and distorted S1 pocket with a buried conformation of Asp189, which is critical for P1 Arg substrate recognition. The FXII structures define patches of negative charge surrounding the active site cleft that may be critical for interactions with inhibitors and substrates.ConclusionsThese data provide the first structural basis for understanding FXII substrate recognition and zymogen activation.

Toward the discovery of dual inhibitors for botulinum neurotoxin A: concomitant targeting of endocytosis and light chain protease activity.

Dyngo-4a™ has been found to be an endocytic inhibitor of BoNT/A neurotoxicity through dynamin inhibition. Herein, we demonstrate this molecule to have a previously unrecognized dual activity against BoNT/A, dynamin-protease inhibition. To establish the importance of this dual activity, detailed kinetic analysis of Dyngo-4a's inhibition of BoNT/A metalloprotease as well as cellular and animal toxicity studies have been described. The research presented is the first polypharmacological approach to counteract BoNT/A intoxication.

A graphene oxide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of botulinum neurotoxin A (BoNT/A) enzymatic activity

Botulinum neurotoxins (BoNTs) are among the most potent toxic bacterial proteins for humans, which make them potential agents for bioterrorism. Therefore, an ultrasensitive detection of BoNTs and their active states is in great need as field-deployable systems for anti-terrorism applications. We report the construction of a novel graphene oxide (GO)-peptide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of the BoNT serotype A light chain (BoNT-LcA) protease activity. A green fluorescence protein (GFP) modified SNAP-25 peptide substrate (SNAP-25-GFP) was optimally designed and synthesized with the centralized recognition/cleavage sites. This FRET platform was constructed by covalent immobilization of peptide substrate on GO with BSA passivation which have advantages of low non-specific adsorption and high stability in protein abundant solution. BoNT-LcA can specifically cleave SNAP-25-GFP substrate covalently immobilized on GO to release the fragment with GFP. Based on fluorescence signal recovery measurement, the target BoNT-LcA was detected sensitively and selectively with the linear detection range from 1fg/mL to 1pg/mL. The limit of detection (LOD) for BoNT-LcA is around 1fg/mL.

Targeting Botulinum A Cellular Toxicity: A Prodrug Approach

The botulinum neurotoxin light chain (LC) protease has become an important therapeutic target for postexposure treatment of botulism. Hydroxamic acid based small molecules have proven to be potent inhibitors of LC/A with nanomolar Ki values, yet they lack cellular activity conceivably due to low membrane permeability. To overcome this potential liability, we investigated two prodrug strategies, 1,4,2-dioxazole and carbamate, based on our 1-adamantylacetohydroxamic acid scaffold. The 1,4,2-dioxazole prodrug did not demonstrate cellular activity, however, carbamates exhibited cellular potency with the most active compound displaying an EC50 value of 20 μM. Cellular trafficking studies were conducted using a "fluorescently silent" prodrug that remained in this state until cellular uptake was complete, which allowed for visualization of the drug's release inside neuronal cells. In sum, this research sets the stage for future studies leveraging the specific targeting and delivery of these prodrugs, as well as other antibotulinum agents, into neuronal cells.

Nanoporous membrane based impedance sensors to detect the enzymatic activity of botulinum neurotoxin A.

Botulinum neurotoxins are among the most potent toxic bacterial proteins for humans and there is a great need to develop simple, rapid and sensitive methods for toxin detection and protease activity quantification in field deployment. In this paper, a nanoporous membrane based impedance sensor was developed to monitor the activity of the BoNT serotype A light chain protease (LcA). Synaptosomal-associated protein 25 (SNAP-25) was first immobilized inside nanopore walls via silane linkers. BoNT LcA was then injected over the nanoporous membrane substrate sensor and specifically cleaved SNAP-25. The cleavage activity could be monitored by measuring impedance signals across nanoporous membranes which represented the nanopore blockage degree. This initial device could achieve a 500 pM LcA detection limit within 25 minutes.

Evaluation of adamantane hydroxamates as botulinum neurotoxin inhibitors: Synthesis, crystallography, modeling, kinetic and cellular based studies

Botulinum neurotoxins (BoNTs) are the most lethal biotoxins known to mankind and are responsible for the neuroparalytic disease botulism. Current treatments for botulinum poisoning are all protein based and thus have a limited window of treatment opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a therapeutic strategy for the treatment of botulism as it may provide an effective post exposure remedy. Using a combination of crystallographic and modeling studies a series of hydroxamates derived from 1-adamantylacetohydroxamic acid (3a) were prepared. From this group of compounds, an improved potency of about 17-fold was observed for two derivatives. Detailed mechanistic studies on these structures revealed a competitive inhibition model, with a Ki=27nM, which makes these compounds some of the most potent small molecule, non-peptidic BoNT/A LC inhibitors reported to date.

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC50 values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).

Synthesis and evaluation of library of betulin derivatives against the botulinum neurotoxin A protease

Botulinum neurotoxins (BoNTs) are the most toxic proteins currently known. Current treatments for botulinum poisoning are all protein based with a limited window of opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a new therapeutic strategy for the treatment of botulism as it may provide an effective post-exposure remedy. As such, a small library of 40 betulin derivatives was synthesized and screened against the light chain of BoNT serotype A (LC/A); five positive hits (IC(50) <100 μM) were uncovered. Detailed evaluation of inhibition mechanism of three most active compounds revealed a competitive model, with sub-micromolar K(i) value for the best inhibitor (7). Unfortunately, an in vitro cell-based assay did not show any protection of rat cerebellar neurons against BoNT/A intoxication by 7.

Monitoring Botulinum Neurotoxin A Activity with Peptide-Functionalized Quantum Dot Resonance Energy Transfer Sensors

Botulinum neurotoxins (BoNTs) are extremely potent bacterial toxins that contaminate food supplies along with having a high potential for exploitation as bioterrorism agents. There is a continuing need to rapidly and sensitively detect exposure to these toxins and to verify their active state, as the latter directly affects diagnosis and helps provide effective treatments. We investigate the use of semiconductor quantum dot (QD)-peptide Förster resonance energy transfer (FRET) assemblies to monitor the activity of the BoNT serotype A light chain protease (LcA). A modular LcA peptide substrate was designed and optimized to contain a central LcA recognition/cleavage region, a unique residue to allow labeling with a Cy3 acceptor dye, an extended linker-spacer sequence, and a terminal oligohistidine that allows for final ratiometric peptide-QD-self-assembly. A number of different QD materials displaying charged or PEGylated surface-coatings were evaluated for their ability to self-assemble dye-labeled LcA peptide substrates by monitoring FRET interactions. Proteolytic assays were performed utilizing either a direct peptide-on-QD format or alternatively an indirect pre-exposure of peptide to LcA prior to QD assembly. Variable activities were obtained depending on QD materials and formats used with the most sensitive pre-exposure assay result demonstrating a 350 pM LcA limit of detection. Modeling the various QD-peptide sensor constructs provided insight into how the resulting assembly architecture influenced LcA recognition interactions and subsequent activity. These results also highlight the unique roles that both peptide design and QD features, especially surface-capping agents, contribute to overall sensor activity.

Iterative Structure-Based Peptide-Like Inhibitor Design against the Botulinum Neurotoxin Serotype A

The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their Ki values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2′ position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.

Identification of a Natural Product Antagonist against the Botulinum Neurotoxin Light Chain Protease

Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNTs are the most lethal known poisons affecting humans and has been recognized as a potential bioterrorist threat. Current treatments for botulinum poisoning are predominately prophylactic in nature relying on passive immunization with antitoxins. Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective post-exposure remedy. A high-throughput screening effort against the light chain of BoNT serotype A (LC/A) was conducted with the John Hopkins Clinical Compound Library comprised of over 1,500 existing drugs. Lomofungin, a natural product first isolated in the late 1960's, was identified as an inhibitor of LC/A, displaying classical noncompetitive inhibition kinetics with a K(i) of 6.7 ± 0.7 µM. Inhibitor combination studies reveal that lomofungin binding is nonmutually exclusive (synergistic). The inhibition profile of lomofungin has been delineated by the use of both an active site inhibitor, 2,4-dichlorocinnamic hydroxamate, and a noncompetitive inhibitor d-chicoric acid; the mechanistic implications of these observations are discussed. Lastly, cellular efficacy was investigated using a rat primary cell model which demonstrated that lomofungin can protect against SNAP-25 cleavage, the intracellular protein target of LC/A.