Small molecule non-peptide inhibitors of botulinum neurotoxin serotype E: Structure–activity relationship and a pharmacophore model

Botulinum neurotoxins (BoNTs) are the most poisonous biological substance known to humans. They cause flaccid paralysis by blocking the release of acetylcholine at the neuromuscular junction. Here, we report a number of small molecule non-peptide inhibitors of BoNT serotype E. The structure–activity relationship and a pharmacophore model are presented. Although non-peptidic in nature, these inhibitors mimic key features of the uncleavable substrate peptide Arg-Ile-Met-Glu (RIME) of the SNAP-25 protein. Among the compounds tested, most of the potent inhibitors bear a zinc-chelating moiety connected to a hydrophobic and aromatic moiety through a carboxyl or amide linker. All of them show low micromolar IC₅₀ values.     

Synthesis and activity of isoleucine sulfonamide derivatives as novel botulinum neurotoxin serotype A light chain inhibitors

The botulinum neurotoxin (BoNT) is the most lethal protein known to man causing the deadly disease botulinum. The neurotoxin, composed of a heavy (HC) and light (LC) chain, work in concert to cause muscle paralysis. A therapeutic strategy to treat individuals infected with the neurotoxin is inhibiting the catalytic activity of the BoNT LC. We report the synthesis, inhibition study and computational docking analysis of novel small molecule BoNT/A LC inhibitors. A structure activity relationship study resulted in the discovery of d-isoleucine functionalized with a hydroxamic acid on the C-terminal and a biphenyl with chlorine at C- 2 connected by a sulfonamide linker at the N-terminus. This compound has a measured IC₅₀ of 0.587 µM for the BoNT/A LC. Computational docking analysis indicates the sulfonamide linker adopts a geometry that is advantageous for binding to the BoNT LC active site. In addition, Arg363 is predicted to be involved in key binding interactions with the scaffold in this study.     

Molecular architecture of botulinum neurotoxin E revealed by single particle electron microscopy

Clostridial botulinum neurotoxin (BoNT) causes a neuroparalytic condition recognized as botulism by arresting synaptic vesicle exocytosis. Although the crystal structures of full-length BoNT/A and BoNT/B holotoxins are known, the molecular architecture of the five other serotypes remains elusive. Here, we present the structures of BoNT/A and BoNT/E using single particle electron microscopy. Labeling of the particles with three different monoclonal antibodies raised against BoNT/E revealed the positions of their epitopes in the electron microscopy structure, thereby identifying the three hallmark domains of BoNT (protease, translocation, and receptor binding). Correspondingly, these antibodies selectively inhibit BoNT translocation activity as detected using a single molecule assay. The global structure of BoNT/E is strikingly different from that of BoNT/A despite strong sequence similarity. We postulate that the unique architecture of functionally conserved modules underlies the distinguishing attributes of BoNT/E and contributes to differences with BoNT/A.     

Botulinum Neurotoxins B and E Translocate at Different Rates and Exhibit Divergent Responses to GT1b and Low pH

Botulinum neurotoxins (BoNTs, serotypes A-G) are the most deadly substances known. Here, we investigated how BoNT/E, a serotype that causes human botulism, translocates into the cytosol of neurons. Analogous to BoNT/B, BoNT/E required binding of the coreceptor, GT1b, to undergo significant secondary structural changes and transform into a hydrophobic protein at low pH. These data indicate that both serotypes act as coincidence detectors for both GT1b and low pH, to undergo translocation. However, BoNT/E translocated much more rapidly than BoNT/B. Also, BoNT/E required only GT1b, and not low pH, to oligomerize, whereas BoNT/B required both. In further contrast to the case of BoNT/B, low pH alone altered the secondary structure of BoNT/E to some degree and resulted in its premature inactivation. Hence, comparison of two BoNT serotypes revealed that these agents exhibit both convergent and divergent responses to receptor interactions, and pH, in the translocation pathway.     

Novel small molecule inhibitors of botulinum neurotoxin A metalloprotease activity

Botulinum neurotoxins (BoNTs) are among the most lethal biological substances to have been weaponized and are listed as biodefense category A agents. Currently, no small molecule (non-peptidic) therapeutics exist to counter this threat; hence, identifying and developing compounds that inhibit BoNTs is a high priority. In the present study, a high-throughput assay was used to identify small molecules that inhibit the metalloprotease activity of BoNT serotype A light chain (BoNT/A LC). All inhibitors were further verified using a HPLC-based assay. Conformational analyses of these compounds, in conjunction with molecular docking studies, were used to predict structural features that contribute to inhibitor binding and potency. Based on these results, a common pharmacophore for BoNT/A LC inhibitors is proposed. This is the first study to report small molecules (non-peptidics) that inhibit BoNT/A LC metalloprotease activity in the low microM range.     

Glycosylated SV2A and SV2B mediate the entry of botulinum neurotoxin E into neurons

Botulinum neurotoxin E (BoNT/E) can cause paralysis in humans and animals by blocking neurotransmitter release from presynaptic nerve terminals. How this toxin targets and enters neurons is not known. Here we identified two isoforms of the synaptic vesicle protein SV2, SV2A and SV2B, as the protein receptors for BoNT/E. BoNT/E failed to enter neurons cultured from SV2A/B knockout mice; entry was restored by expressing SV2A or SV2B, but not SV2C. Mice lacking SV2B displayed reduced sensitivity to BoNT/E. The fourth luminal domain of SV2A or SV2B alone, expressed in chimeric receptors by replacing the extracellular domain of the low-density lipoprotein receptor, can restore the binding and entry of BoNT/E into neurons lacking SV2A/B. Furthermore, we found disruption of a N-glycosylation site (N573Q) within the fourth luminal domain of SV2A rendered the mutant unable to mediate the entry of BoNT/E and also reduced the entry of BoNT/A. Finally, we demonstrate that BoNT/E failed to bind and enter ganglioside-deficient neurons; entry was rescued by loading exogenous gangliosides into neuronal membranes. Together, the data reported here demonstrate that glycosylated SV2A and SV2B act in conjunction with gangliosides to mediate the entry of BoNT/E into neurons.     

Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.     

The receptor binding domain of botulinum neurotoxin serotype C binds phosphoinositides

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD₅₀ of ∼1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a “dual receptor” mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro domain. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides in both assays. Interactions with phosphoinositides may facilitate tighter binding between neuronal membranes and BoNT/C.     

Conformational analysis of GT1B ganglioside and its interaction with botulinum neurotoxin type B: a study by molecular modeling and molecular dynamics

The conformational property of oligosaccharide GT1B in aqueous environment was studied by molecular dynamics (MD) simulation using all-atom model. Based on the trajectory analysis, three prominent conformational models were proposed for GT1B. Direct and water-mediated hydrogen bonding interactions stabilize these structures. The molecular modeling and 15 ns MD simulation of the Botulinum Neuro Toxin/B (BoNT/B) - GT1B complex revealed that BoNT/B can accommodate the GT1B in the single binding mode. Least mobility was seen for oligo-GT1B in the binding pocket. The bound conformation of GT1B obtained from the MD simulation of the BoNT/B-GT1B complex bear a close conformational similarity with the crystal structure of BoNT/A-GT1B complex. The mobility noticed for Arg 1268 in the dynamics was accounted for its favorable interaction with terminal NeuNAc. The internal NeuNAc1 tends to form 10 hydrogen bonds with BoNT/B, hence specifying this particular site as a crucial space for the therapeutic design that can restrict the pathogenic activity of BoNT/B.     

Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C–D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (∼two-third) and BoNT/D (∼one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.     

Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E

BackgroundBotulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food-poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure.ResultsIn this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD₅₀/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD₅₀ BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay.ConclusionThese scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development.     

Benzoquinones as inhibitors of botulinum neurotoxin serotype A

Although botulinum neurotoxin serotype A (BoNT/A) is known for its use in cosmetics, it causes a potentially fatal illness, botulism, and can be used as a bioterror weapon. Many compounds have been developed that inhibit the BoNTA zinc-metalloprotease light chain (LC), however, none of these inhibitors have advanced to clinical trials. In this study, a fragment-based approach was implemented to develop novel covalent inhibitors of BoNT/A LC. First, electrophilic fragments were screened against BoNT/A LC, and benzoquinone (BQ) derivatives were found to be active. In kinetic studies, BQ compounds acted as irreversible inhibitors that presumably covalently modify cysteine 165 of BoNT/A LC. Although most BQ derivatives were highly reactive toward glutathione in vitro, a few compounds such as natural product naphthazarin displayed low thiol reactivity and good BoNT/A inhibition. In order to increase the potency of the BQ fragment, computational docking studies were employed to elucidate a scaffold that could bind to sites adjacent to Cys165 while positioning a BQ fragment at Cys165 for covalent modification; 2-amino-N-arylacetamides met these criteria and when linked to BQ displayed at least a 20-fold increase in activity to low μM IC₅₀ values. Unlike BQ alone, the linked-BQ compounds demonstrated only weak irreversible inhibition and therefore acted mainly as non-covalent inhibitors. Further kinetic studies revealed a mutual exclusivity of BQ covalent inactivation and competitive inhibitor binding to sites adjacent to Cys165, refuting the viability of the current strategy for developing more potent irreversible BoNT/A inhibitors. The highlights of this study include the discovery of BQ compounds as irreversible BoNT/A inhibitors and the rational design of low μM IC₅₀ competitive inhibitors that depend on the BQ moiety for activity.     

Discovery of a series of small molecules as potent histone deacetylase inhibitors

Abstract

A series of small molecules were designed and synthesized based on our previous virtual screening approach, which was performed to discover potent histone deacetylase inhibitors (HDACIs) with novel structures. The derived compounds were tested by Hela cell nucleus extract for enzyme inhibition assay. Tumor cell growth inhibition assays were performed using a series of tumor cell lines. Molecule 4h has the best performance among these compounds with enzyme inhibition IC₅₀ of 0.14?μM and tumor cell growth inhibition IC₅₀ of 1.85 (U937), 2.02 (HL60), 2.67 (K562). Docking studies showed that multiple H-bonds and hydrophobic interactions make 4h binding to the active site of HDAC. 4h has the advantage of low molecular weight, so a variety of structural modifications can be performed in our further studies.     

SNAP-25 is expressed in islets of Langerhans and is involved in insulin release

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP- 25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.     

Molecular Dynamics Simulations of Sonic Hedgehog-Receptor and Inhibitor Complexes and Their Applications for Potential Anticancer Agent Discovery

The sonic hedgehog (Shh) signaling pathway is necessary for a variety of development and differentiation during embryogenesis as well as maintenance and renascence of diverse adult tissues. However, an abnormal activation of the signaling pathway is related to various cancers. In this pathway, the Shh signaling transduction is facilitated by binding of Shh to its receptor protein, Ptch. In this study, we modeled the 3D structure of functionally important key loop peptides of Ptch based on homologous proteins. Using this loop model, the molecular interactions between the structural components present in the pseudo-active site of Shh and key residues of Ptch was investigated in atomic level through molecular dynamics (MD) simulations. For the purpose of developing inhibitor candidates of the Shh signaling pathway, the Shh pseudo-active site of this interface region was selected as a target to block the direct binding between Shh and Ptch. Two different structure-based pharmacophore models were generated considering the key loop of Ptch and known inhibitor-induced conformational changes of the Shh through MD simulations. Finally two hit compounds were retrieved through a series of virtual screening combined with molecular docking simulations and we propose two hit compounds as potential inhibitory lead candidates to block the Shh signaling pathway based on their strong interactions to receptor or inhibitor induced conformations of the Shh.     

Glycine insertion at protease cleavage site of SNAP25 resists cleavage but enhances affinity for botulinum neurotoxin serotype A

The light chain of botulinum neurotoxin A (BoNT/A‐LC) is a Zn‐dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C‐terminus of SNAP25 (residues 141–206) retains full activity for BoNT/A‐LC‐catalyzed cleavage at P1‐P1' (Gln197‐Arg198). Using the structure of a complex between the C‐terminus of SNAP25 and BoNT/A‐LC as a model to design SNAP25‐derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala‐Asn‐Gln‐Arg (residues 195–198) at the substrate cleavage site, with the intent to identify possible side‐chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1‐P1' residues to explore the spatial tolerance within the active‐site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A‐LC. Among the SNAPIs tested, several known cleavage‐resistant, single‐point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly‐insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A‐LC. Two of the Gly‐insertion mutants exhibited 10‐fold lower IC₅₀ values than the wildtype 66‐mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.     

Scoring ligand similarity in structure‐based virtual screening

Scoring to identify high‐affinity compounds remains a challenge in virtual screening. On one hand, protein–ligand scoring focuses on weighting favorable and unfavorable interactions between the two molecules. Ligand‐based scoring, on the other hand, focuses on how well the shape and chemistry of each ligand candidate overlay on a three‐dimensional reference ligand. Our hypothesis is that a hybrid approach, using ligand‐based scoring to rank dockings selected by protein–ligand scoring, can ensure that high‐ranking molecules mimic the shape and chemistry of a known ligand while also complementing the binding site. Results from applying this approach to screen nearly 70 000 National Cancer Institute (NCI) compounds for thrombin inhibitors tend to support the hypothesis. EON ligand‐based ranking of docked molecules yielded the majority (4/5) of newly discovered, low to mid‐micromolar inhibitors from a panel of 27 assayed compounds, whereas ranking docked compounds by protein–ligand scoring alone resulted in one new inhibitor. Since the results depend on the choice of scoring function, an analysis of properties was performed on the top‐scoring docked compounds according to five different protein–ligand scoring functions, plus EON scoring using three different reference compounds. The results indicate that the choice of scoring function, even among scoring functions measuring the same types of interactions, can have an unexpectedly large effect on which compounds are chosen from screening. Furthermore, there was almost no overlap between the top‐scoring compounds from protein–ligand versus ligand‐based scoring, indicating the two approaches provide complementary information. Matchprint analysis, a new addition to the SLIDE (Screening Ligands by Induced‐fit Docking, Efficiently) screening toolset, facilitated comparison of docked molecules' interactions with those of known inhibitors. The majority of interactions conserved among top‐scoring compounds for a given scoring function, and from the different scoring functions, proved to be conserved interactions in known inhibitors. This was particularly true in the S1 pocket, which was occupied by all the docked compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

Multiple pocket recognition of SNAP25 by botulinum neurotoxin serotype E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. The molecular basis for SNAP25 recognition and cleavage by BoNT serotype E is currently unclear. Here we define the multiple pocket recognition of SNAP25 by LC/E. The initial recognition of SNAP25 is mediated by the binding of the B region of SNAP25 to the substrate-binding (B) region of LC/E comprising Leu166, Arg167, Asp127, Ala128, Ser129, and Ala130. The mutations at these residues affected substrate binding and catalysis. Three additional residues participate in scissile bond cleavage of SNAP25 by LC/E. The P3 site residues, Ile178, of SNAP25 interacted with the S3 pocket in LC/E through hydrophobic interactions. The S3 pocket included Ile47, Ile164, and Ile182 and appeared to align the P1' and P2 residues of SNAP25 with the S1' and S2 pockets of LC/E. The S1' pocket of LC/E included three residues, Phe191, Thr159, and Thr208, which contribute hydrophobic and steric interactions with the SNAP25 P1' residue Ile181. The S2 pocket residue of LC/E, Lys224, binds the P2 residue of SNAP25, Asp179, through ionic interactions. Deletion mapping indicates that main chain interaction(s) of residues 182-186 of SNAP25 contribute to substrate recognition by LC/E. Understanding the mechanism for substrate specificity provides insight for the development of inhibitors against the botulinum neurotoxins.     

Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep–MS assay

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD₅₀/ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples.     

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 (IC₅₀ <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.