Development of an antibody-based screen for the identification of novel acetyl coenzyme-A carboxylase inhibitors.

摘要

The cyclohexanediones are one of four known structural classes of herbicides that inhibit acetyl coenzyme-A carboxylase (ACCase), the first step in fatty acid biosynthesis. Research was conducted to develop ACCase inhibitor-specific antibodies and evaluate them as screening tools to identify novel lead chemistry. Both polyclonal and monoclonal antibodies were produced against a cyclohexanedione hapten conjugated to bovine serum albumin.; Two competitive, indirect enzyme-linked immunosorbent assays (ciELISA) using two cyclohexanedione haptens conjugated to ovalbumin as coating conjugates were developed using the polyclonal antiserum. Nineteen cyclohexanedione analogues, 13 active ACCase inhibitors and 6 inactive analogues were tested for their ability to compete with both coating conjugates for antiserum binding. All active ACCase inhibitors were observed to compete with both coating conjugates, whereas, all inactive analogues failed to compete with at least one coating conjugate in the two ciELISAs. To improve the specificity of the screen, monoclonal antibodies were produced against the cyclohexanedione immunogen.; A total of five monoclonal antibodies were produced and cross-reactivity studies using the homologous ciELISA (same cyclohexanedione hapten used for the immunogen and coating conjugate) against a total of 24 cyclohexanedione analogues revealed two monoclonal antibodies (mAbA and mAbB) could segregate the analogues into active and non-active ACCase inhibitors on the basis of analogue concentration required to inhibit 50% of monoclonal antibody binding to the coating conjugate (IC50). Both monoclonal antibodies also cross-reacted with analogues from the indolizine-2,4-dione structural class of ACCase inhibitors. However, the current homologous ciELISA lacked the sensitivity required to identify all known structural classes of ACCase inhibitors.; To enhance the sensitivity of the monoclonal antibody based screen two additional heterologous ciELISAs were developed with coating conjugates based on non-active cyclohexanedione haptens. Monoclonal antibody cross-reactivity with the cyclohexanedione, aryloxyphenoxypropanoic acid, indolizine-2,4-dione, and triazinedione structural classes of ACCase inhibitors was determined in the heterologous ciELISAs. These studies revealed mAbA could cross-react with active analogues from all four structural classes of ACCase inhibitors tested. Furthermore, mAbA was used as a surrogate target site in a pilot screen where two novel ACCase inhibitors were identified.; To better understand the relationship between the antibody and enzyme the three-dimensional quantitative structure activity comparative molecular field analysis (3D-QSAR CoMFA) methodology was used to identify similarities and differences between the steric and electrostatic potential fields of mAbA and ACCase. Two separate CoMFA models were developed to explain the interaction of cyclohexanedione analogues with ACCase and mAbA. Similarities in the steric and electrostatic potential graphs around the position 2 oxime functional group of the cyclohexanedione molecule can account for the qualitative relationship observed for cyclohexanedione inhibition of enzyme activity and antibody binding. The resulting CoMFA models indicate both mAbA and ACCase have some similar steric and electrostatic requirements for interaction with the cyclohexanediones. These results suggest surrogate antibody target site mimics with steric and electrostatic potentials comparable to the natural target site can be produced and these antibody target site mimics can be used as screening tools in pesticide discovery programs.