Metabolism of cydiastatin 4 and analogues by enzymes associated with the midgut and haemolymph of Manduca sexta larvae

Authors: AUDSLEY, NEIL - CENTRAL SCIENCE LAB, UK; MATTHEWS, JUNE - CENTRAL SCIENCE LAB, UK; Nachman, Ronald; WEAVER, ROBERT - CENTRAL SCIENCE LAB, UK

Submitted to: General and Comparative Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/13/2007
Publication Date: 2/20/2007
Citation: Audsley, N., Matthews, J., Nachman, R.J., Weaver, R.J. 2007. Metabolism of cydiastatin 4 and analogues by enzymes associated with the midgut and haemolymph of Manduca sexta larvae. General and Comparative Endocrinology. 153:80-87.

Interpretive Summary: Because of problems with the development of resistance to conventional pesticides, there is a critical need for new concepts and alternative approaches in controlling insect pests. The basic premise of this research is that neuropeptides (short chains of amino acids) serve as potent messengers in insects to regulate vital functions. Nevertheless, neuropeptides in and of themselves hold little promise as pest control agents because of susceptibility to being degraded in the target pest. Neuropeptide mimics must be designed that resist degradation by enzymes in the digestive tract and blood of pest insects and interact with the active site within agricultural or medical pests by over-activating or blocking critical, neuropeptide-regulated life functions. We report on the characterization of pathways in the tobacco hawkmoth involved in the degradation of the cydiastatin class of neuropeptides, implicated in regulation of developmental and reproductive processes, and the design and evaluation of a cydiastatin mimic. The mimic demonstrated greatly enhanced stability to enzymes that degrade the natural regulatory neuropeptide. This work represents an important milestone and lead in the development of practical neuropeptide-like substances that will effectively control insect pests in an environmentally friendly fashion.

Technical Abstract: The degradation of synthetic cydiastatin 4 (ARPYSFGL-amide) and cydiastatin 4 analogues cydiastatin 4a (PPPPPARPYSFGL-amide) and cydiastatin 4b (PPPPPARPYSF[Acpc]L-amide) by enzymes associated with the midgut and/or haemolymph of the tobacco hawkmoth moth, Manduca sexta were investigated using reversed-phase high performance liquid chromatography (RP-HPLC) combined with matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). Cydiastatin 4 had an estimated half-life of c. 16.5 minutes when incubated with midgut tissue in vitro and c. 2.5 minutes with midgut lumen contents. Two degradation products were identified; cydiastatin, due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin, due to cleavage of the N-terminal A residue. Both cydiastatin 4a and cydiastatin 4b had increased stability to gut and haemolymph enzymes, and full biological activity, but reduced potency compared to cydiastatin 4 when assayed on foregut peristalsis. The P-extended N-terminus of both analogues prevented hydrolysis by aminopeptidases and the replacement of the susceptible G residue with cyclopropylalanine ([Acpc]) counteracted carboxypeptidase activity. However, both analogues were susceptible to amidase-like activity giving an increase in one mass unit presumably due to the conversion of the C-terminal amide group to the free carboxylic acid. No metabolism of cydiastatin 4b occurred when incubated with larval M. sexta haemolymph over a 90-minute period.