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determine if they could retain the fluid secretion stimulatory effects of the natural insectkinins and parent APy analogs. Despite the major structural modifications present in thesemimetic analogs in comparison with the natural insect kinins and even the parent APyanalogs, the two biostable analogs retained significant diuretic activity on the cricketMalpighian tubule fluid secretion assay. Both retained activity at a threshold concentrationbetween 1 and 10 μM; and while the activity of 1797 appeared to be somewhat greater thanthat of 1796, this difference was not statistically significant under these experimentalconditions. For comparison, native achetakinin I (AK-I) at 1 μM is also included inFigure 2. The maximal response (efficacy) of the two analogs and the natural AK-I arestatistically equivalent.% increase in secretion10075502501796 1797 AKI10 M1 MFig. 2. Diuretic activity of mimetic insect kinin analog 1796 and 1797 compared withnative AK-I.The two peptidomimetic APy analogs of the insect kinin C-terminal pentapeptide activecore feature major structural modifications in comparison with the natural sequence,including subsititution of the Phe 1 residue with Hca, replacement of the peptide linkagebetween the C-terminal Trp 4 -Gly 5 residue block with an isosteric reduced bond and a cyclicAPy linkage as a replacement for the Xaa 2 -Pro 3 dipeptide block. As a consequence, theanalogs contain no native peptide linkages that would be expected to render themsusceptible to hydrolysis by either exo- or endo-peptidases in the hemolymph (blood) ortissues of pest insects. A diuretic agonist that the target insect is unable to inactivate vianormal proteolytic pathways offers the potential to disrupt the water balance critical forinsect survival. Previous reports indicate that while other insect kinin analogs withenhanced biostability show potency levels that are several orders of magnitude less thannative peptides in in vitro Malpighian tubule secretion assays they nonetheless match thepotency of the native peptides in an in vivo housefly diuretic assay [1-3]. Interestingly,biostable insect kinin analogs containing Aib residues have recently been reported todemonstrate potent oral aphicidal effects [4].The active biostable insect kinin analogs described in this study and/or 2nd generationanalogs, either in isolation or in combination with biostable analogs of other neuropeptideclasses that also regulate aspects of diuretic, antidiuretic, digestive, reproductive and/ordevelopmental processes, represent potential leads in the development of selective,environmentally friendly pest insect control agents capable of disrupting those criticalprocesses.AcknowledgmentsWe thank Allison Strey and Nan Pryor for technical assistance. We acknowledge financial assistancefrom the North Atlantic Treaty Organization (NATO) Collaborative Research Grant(#LST.CLG.979226) and the USDA/DOD DWFP Research Initiative (#00500-32000-001-01R).References1. Nachman, R.J., Zabrocki, J., Olczak, J., Williams, H.J., Moyna, G., Scott, A.I., Coast, G.M. Peptides23, 709-745 (2002).2. Nachman, R.J., Kaczmarek, K., Williams, H.J., Coast, G.M., Zabrocki, J. Biopolymers 75, 412-419(2004).3. Kaczmarek, K., Williams, H.J., Coast, G.M., Scott, A.I., Zabroki, J., Nachman, R.J. Peptide Science88, 1-7 (2006).4. Smagghe, G., Mahdian, K., Zubrzak, P., Nachman, R.J. Peptides 31, 498-505 (2010).423