High level expression and immunochemical characterization of botulinum neurotoxin type F light chain.

Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis.

In Silico Discovery and Validation of Amide Based Small Molecule Targeting the Enzymatic Site of Shiga Toxin.

Shiga toxin (Stx), a category B biothreat agent, is a ribosome inactivating protein and toxic to human and animals. Here, we designed and synthesized small molecules that block the active site of the Stx A subunit. On the basis of binding energy, 20 molecules were selected for synthesis and evaluation. These molecules were primarily screened using fluorescence-based thermal shift assay and in vitro in Vero cells. Among 32 molecules (including 12 reported), six molecules offered protection with IC50 of 2.60-23.90 µM. 4-Nitro-N-[2-(2-phenylsulfanylethylamino)ethyl]benzamide hydrochloride is the most potent inhibitor with IC50 at 7.96 µM and selectivity index of 22.23 and is better than any known small molecule inhibitor of Stx. Preincubation with Stx offered full protection against Shiga toxin in mice. Surface plasmon resonance assay further confirmed that these molecules bind specifically to Stx A subunit. Further optimization is continued to identify a potential candidate which will be in vivo effective.

Expression, Purification and Immunological Characterization of Recombinant Shiga Toxin A Subunit.

Shiga toxin family comprises toxins belonging to two major groups, Stx1 and Stx2, produced by the bacteria Shigella dysenteriae and some strains of Escherichia coli. Shiga toxins are the leading cause of diarrhea associated with life threatening hemolytic uremic syndrome (HUS). StxA is a ribosome inactivating protein (RIP) which inhibits the protein synthesis in most species of prokaryotes and eukaryotes. An in vitro expression system has not been reported to produce full-length biological active StxA subunit; hence substantial progress has been hampered. In the present study, a DNA fragment (955 bp Gene Bank Accn No HM017965) encoding for subunit A of Stx was amplified from Shigella dysenteriae type 1 and subsequently cloned in pGEX-5X-2 vector. We successfully produced recombinant StxA as GST fusion protein in Escherichia coli using pGEX-5X-2-STXA construct under IPTG induction. For the purpose of immunization the GST-tag was removed by factor Xa mediated endoproteolytic cleavage from GST-StxA. Antisera raised against rStxA in mice reacted with recombinant purified protein of rStxA and lysate of Shiga toxin. It was shown that antisera produced against rStxA significantly recognized Stx producing strains of S. dysenteriae and E. coli. The antiserum produced effectively neutralized the Shiga toxin's cytotoxicity towards Vero cells.

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 IC(50) 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).