DTIC ADA500949: Structure-Activity Relationships for in vitro Diuretic Activity of CAP2b in the Housefly

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peptides 28 (2007) 57–61
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journal homepage: www.elsevier.com/locate/peptides
Structure-activity relationships for in vitro diuretic
activity of CAP2b in the housefly
Ronald J. Nachmana,*, Geoffrey M. Coastb
aAreawidePestManagementResearch,SouthernPlainsAgriculturalResearchCenter,USDA,2881F/BRoad,
CollegeStation,TX77845,USA
bSchoolofBiologicalandChemicalSciences,Birkbeck(UniversityofLondon),LondonWC1E7HX,UK
article info a bs t r ac t
Articlehistory: A series of truncated and Ala-replacement analogs of the peptide Manse-CAP2b
Received11September2006 (pELYAFPRV-NH )wereassayedfordiureticactivityonMalpighiantubulesofthehousefly
2
Receivedinrevisedform Muscadomestica(M.domestica).TheC-terminalhexapeptideprovedtobetheactivecore,the
15September2006 minimumsequencerequiredtoretainsignificantdiureticactivity.However,fullactivity
Accepted15September2006 requiredtheC-terminalheptapeptide,whichwasequipotentwiththemostactiveofthe
Publishedonline8December2006 nativehouseflyCAP2bpeptides.ReplacementofArg7andVal8withAlaledtoinactivity
and a large 70-fold drop in potency, respectively, indicating that these were critical
Keywords: residues. The Leu2 was semicritical, where a six-fold loss in potency was observed.
CAPAgene Conversely,thereplacementofallotherresidueswithAlaledtomuchsmallereffectson
Periviscerokinin potency and these positions were considered to be noncritical. This structure-activity
CAP2b relationship data can aid in the design of mimetic agonist/antagonist analogs of this
Perisympatheticorgans diuretic peptide family with enhanced biostability and bioavailability, as tools for
Insectneuropeptide arthropodendocrinologistsandaspotentialpestmanagementagentscapableofdisrupting
Malpighiantubule thewaterbalanceinpestflies.
#2006ElsevierInc.Allrightsreserved.
1. Introduction bioassays on the bug Rhodnius prolixus with Manduca CAP2b
[23,29] indicated that native CAPA peptides might reduce
It is well established that neuropeptides are key factors in secretion by Malpighian tubules and thus show antidiuretic
controlling primary urine production in Malpighian tubules effects. These peptides are putative hormones typical of the
(CRF-like peptides, insect kinins, CAPA peptides) and fluid neurosecretory system in the abdominal ventral nerve cord
reabsorption from the hindgut (ion transport peptide (ITP)) (VNC)ofinsects.TheCAPAgene,whichencodesfortwoormore
[1,29].CAP2b(pELYAFPRVa),aCAPApeptide(alsoincludedin CAP2b/PVKs and a single pyrokinin (PK), is known from a
the literature as a member of the periviscerokinin (PVK) number of holometabolous insects, including D. melanogaster
family), was first identified as a cardioacceleratory factor in [12,13]andM.sexta[14].Expressedinmedianneurosecretory
themothManducasexta[11].CAPApeptides,includingCAP2b, neurons of abdominal ganglia, these putative peptide hor-
demonstrate diuretic effects on the Malpighian tubules of a mones may be releasedintothe hemolymph via perisympa-
numberofflies,includingthefruitflyDrosophilamelanogaster, thetic organs (PSOs) which are segmentally reiterated
thehouseflyMuscadomestica(M.domestica),andthestablefly neurohemalorgansoftheabdominalganglia[7,8].LarvalPSOs
Stomoxyscalcitrans[1,2–5,9,10,15,18,21,22,24–26,28].Bycontrast, of cycloraphous Diptera, however, become incorporated into
* Correspondingauthor.Tel.:[phone];fax:[phone].
E-mailaddress:[email protected](R.J.Nachman).
0196-9781/$–seefrontmatter#2006ElsevierInc.Allrightsreserved.
doi:10.1016/j.peptides.2006.09.020
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1. REPORT DATE 3. DATES COVERED
15 SEP 2006 2. REPORT TYPE 00-00-2006 to 00-00-2006
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Structure-activity relationships for in vitro diuretic activity of CAP2b in
5b. GRANT NUMBER
the housefly
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6. AUTHOR(S) 5d. PROJECT NUMBER
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7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
U.S. Department of Agriculture,Areawide Pest Management REPORT NUMBER
Research,2881 F/B Road,College Station,TX,77845
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58 peptides 28 (2007) 57–61
thedorsalganglionicsheath[20,30,31]duringmetamorphosis. Manse-CAP2b[4-8], 11.5min; Manse-CAP2b[5-8], 10.5min;
DirectanalysisofabdominaldorsalsheathtissuesviaMALDI- Manse-CAP2b[Ala1], 15.5min; Manse-CAP2b[Ala2], 15.0min;
TOF/TOFmassspectrometryledtotheidentificationofCAP2b/ Manse-CAP2b[Ala3], 13.5min; Manse-CAP2b[Ala5], 16.5min;
PVKpeptidesnativetothehouseflyM.domestica[18,26,32]and Manse-CAP2b[Ala6], 11.0min; Manse-CAP2b[Ala7], 5.5min;
three other species of flies [18,19]. The housefly CAP2b/PVK Manse-CAP2b[Ala8],15.0min.Aminoacidanalysiswascarried
sequenceswererecentlydeterminedtobeAGGTSGLYAFPRVa out under previously reported conditions [17] and used to
(Musdo-CAP2b/PVK-1)andASLFNAPRVa(Musdo-CAP2b/PVK-2) quantify the peptide and to confirm identity, leading to the
[18]. The octapeptide CAP2b shares a common C-terminal following analyses: Manse-CAP2b[2-8]: A[1.0], F[1.0], L[1.0],
heptapeptide sequence (LYAFPRVa) with the most active P[1.0], R[1.0], Y[1.0], V[1.0]; Manse-CAP2b[3-8]: A[1.0], F[1.0],
houseflyCAP2bsequence,Musdo-CAP2b-1,revealingthelikely P[1.1], R[1.0], Y[1.0], V[1.0]; Manse-CAP2b[4-8]: A[0.8], F[1.0],
reasonthattheManducapeptidedemonstratesrelativelypotent P[1.0], R[1.0], V[0.9]; Manse-CAP2b[5-8]: F[1.0], P[1.0], R[1.0],
diureticactivityintheheterologoushouseflyMalpighiantubule V[1.0]; Manse-CAP2b[Ala1]: A[1.9], F[1.0], L[1.0], P[1.0], R[1.0],
fluidsecretionbioasssay. Y[1.1], V[0.9]; Manse-CAP2b[Ala2]: A[2.0], E[0.9], F[1.0], P[0.9],
Inthisstudy,weuseManducaCAP2basamodelofMusdo- R[1.0], Y[1.1], V[1.0]; Manse-CAP2b[Ala3]: A[2.0], E[0.9], F[1.0],
CAP2b-1andevaluateaseriesoftruncatedandAla-replace- L[1.0], P[1.0], R[1.0], V[0.9]; Manse-CAP2b[Ala5]: A[2.0], E[0.8],
ment CAP2b analogs in a housefly Malpighian tubule fluid L[1.0], P[0.9], R[1.0], Y[1.2], V[0.9]; Manse-CAP2b[Ala6]: A[2.0],
secretion bioassay to identify the active core and those E[1.0], F[1.0], L[0.9], R[1.0], Y[1.0], V[1.0]; Manse-CAP2b[Ala7]:
residuesmostcriticaltoactivity. A[2.1], E[0.7], F[1.0], L[1.1], P[1.1], Y[0.8], V[1.0]; Manse-CAP2-
b[Ala8]: A[2.0], E[1.0], F[1.0], L[1.0], P[0.9], R[1.0], Y[1.0]. The
identities of the peptide analogs were confirmed via MALDI-
2. Materials and methods TOF-MSonaKratosKompactProbeMALDI-TOFMSmachine
(KratosAnalytical,Ltd.,Manchester,UK)withthepresenceof
2.1. Insects thefollowingmolecularions(M+H+):Manse-CAP2b[2-8],864.9
[M+H+];Manse-CAP2b[3-8],751.5[M+H+];Manse-CAP2b[4-8],
Housefly(M.domestica)larvaewereobtainedfromacommercial 588.6[M+H+];Manse-CAP2b[5-8],517.6[M+H+];Manse-CAP2-
fishbaitsupplier.Theywereheldinthelaboratoryuntilthey b[Ala1], 936.8 [M+H+]; Manse-CAP2b[Ala2], 933.8 [M+H+];
pupatedandwerethenmovedtoaconstanttemperatureroom Manse-CAP2b[Ala3], 883.9 [M+H+]; Manse-CAP2b[Ala5], 899.5
(288C;12L:12Dlight:darkcycle).Fliesthatemergedwithin12h [M+H+]; Manse-CAP2b[Ala6], 949.3 [M+H+]; Manse-CAP2-
ofoneanotherwerekeptasseparateagecohortsandprovided b[Ala7],890.6[M+H+];Manse-CAP2b[Ala8],947.7[M+H+].
with water and sucrose ad libidum. All of the experiments
describedinthispaperused3–4day-oldadultfemaleflies. 2.3. IsolatedhouseflyMalpighiantubulepreparations
2.2. Peptidesynthesis FluidsecretionfromisolatedhouseflyMalpighiantubuleswas
measuredusingthe‘‘Ramsayassay’’aspreviouslydescribed
TheCAP2banalogsweresynthesizedviaFmocmethodology [10]. Tubules were removed from 3 to 4 day post-emergent
on Rink Amide resin (Novabiochem, San Diego, CA) using adultfemaleflies.FliesweredissectedunderMuscasaline[10]
Fmocprotectedaminoacids(AdvancedChemtech,Louisville, and both anterior and posterior tubules were transferred to
KY)onanABI433Apeptidesynthesizer(AppliedBiosystems, small(10ml)dropsofbathingfluid(a1:1mixtureofsalineand
Foster City, CA) under previously described conditions [17]. Schneider’s Drosophila medium) beneath water-saturated
CrudeproductswerepurifiedonaWatersC SepPakcartridge liquid paraffin in a SylgardTM lined Petri dish. The tubules
18
andaDeltaPakC reverse-phasecolumn(8mm(cid:2)100mm, were allowed to equilibrate for 1h before being challenged
18
15mm particle size, 100A˚ pore size) on a Waters 510 HPLC with test peptides. Urine escaped from the cut end of the
controlledwithaMillennium2010chromatographymanager tubule,whichwaspulledoutintotheliquidparaffin.Dropsof
system (Waters, Milford, MA) with detection at 214nm at urinewerecollectedat15minintervalsandtheirdiameter(d)
ambienttemperature.SolventA=0.1%aqueoustrifluoroace- measuredastheyrestedontheSylgardbaseofthePetridish
ticacid(TFA);SolventB=80%aqueousacetonitrilecontaining using a Wild digital (MMS235) eyepiece micrometer. Urine
0.1%TFA.Conditions:initialsolventconsistingof20%Bwas volume was calculated as pd3/6 and the rate of secretion
followedbytheWaterslinearprogramto100%Bover40min; obtained by dividing the secreted volume by the collection
flow rate, 2.0ml/min. Delta-Pak C-18 retention times: period. Data were normalized by expressing the increase in
Manse-CAP2b[2-8], 9.0min; Manse-CAP2b[3-8], 7.5min; fluid secretion as a percentage of the response to
Manse-CAP2b[4-8], 5.0min; Manse-CAP2b[5-8], 4.5min; 10nMMusdo-K [16], which was added to all tubules at the
Manse-CAP2b[Ala1], 10.5min; Manse-CAP2b[Ala2], 9.0min; endofeachexperiment.Dose-responsecurveswereprepared
Manse-CAP2b[Ala3], 4.5min; Manse-CAP2b[Ala5], 7.5min; using the computer program GraphPad Prism version 4.02
Manse-CAP2b[Ala6], 12.5min; Manse-CAP2b[Ala7], 10.5min; (GraphPadSoftware,SanDiego,CA).
Manse-CAP2b[Ala8], 9.0min. The peptides were further pur-
ified on a Waters Protein Pak I125 column (7.8mm(cid:2)
300mm)(Milligen Corp., Milford, MA). Conditions: flow rate: 3. Results
2.0ml/min;isocraticwithsolventconsistingof80%acetoni-
trile made to 0.01% TFA. WatPro retention times: A list of the fluid secretion activity of the two CAP2b/PVK
Manse-CAP2b[2-8], 7.0min; Manse-CAP2b[3-8], 8.0min; peptidesnativetothehousefly,Manse-CAP2b,Manse-CAP2b
peptides 28 (2007) 57–61 59
Table1–FluidsecretionactivityofCAP2b/PVKpeptidesandanalogsonhousefly(M.domestica)Malpighiantubules
Peptide Sequence Fluidsecretiona
EC (nM)(95%CL) Maximalresponse(%)
50
Musdo-CAP2b/PVK-1 AGGTSGLYAFPRVa 9(7.2–11.1)[6] 100
Musdo-CAP2b/PVK-2 ASLFNAPRVa 102(96–109)[6] 100
Manse-CAP2b pELYAFPRVa 53(39–72)[6]
Manse-CAP2b[2-8] LYAFPRVa 8(3–22) 100
Manse-CAP2b[3-8] YAFPRVa 428(298–613) 100
Manse-CAP2b[4-8] AFPRVa Tr
Manse-CAP2b[5-8] FPRVa Tr
Manse-CAP2b[Ala1] ALYAFPRVa 23(11–46) 100
Manse-CAP2b[Ala2] pEAYAFPRVa 373(255–544) 80
Manse-CAP2b[Ala3] pELAAFPRVa 33(10–105) 100
Manse-CAP2b[Ala5] pELYAAPRVa 115(27–485) 70
Manse-CAP2b[Ala6] pELYAFARVa 89(62–129) 100
Manse-CAP2b[Ala7] pELYAFPAVa Inactive
Manse-CAP2b[Ala8] pELYAFPRAa 3500(2250–5500) 45
a Valuesarederivedfromdose–responsecurvesbasedondatapointsderivedfromsixreplicates.
truncated analogs, and a Manse-CAP2b Ala scan series of 4. Discussion
analogs on housefly Malpighian tubules is presented in
Table 1. The fluid secretion activity of Manduca CAP2b TheevaluationoftheCAP2bsequencefromM.sexta,Manse-
(EC =53nM) lies midway between that of the native CAP2b, in heterologous Malpighian tubule fluid secretion
50
Musdo-CAP2b/PVK-1 (EC =9nM) and Musdo-CAP2b/PVK- assays in fruit flies and houseflies [3,5,18] established that
50
2(EC =102nM);andthuscanserveasareasonablemodel this family of peptides demonstrated diuretic activity in
50
for a structure-activity relationship study of CAP2b/PVK addition to cardioacceleratory activity. Seven of the eight
peptides in the housefly. Interestingly, removal of the pE residuesofManse-CAP2bareidenticalwiththemostpotent
residue on the N-terminus of Manse-CAP2b leads to the C- of the native housefly CAP2b/PVK peptides, Musdo-CAP2b/
terminal heptapeptide sequence common to both Manse- PVK-1. As can be seen in Table 1, the EC for housefly
50
CAP2bandMusdo-CAP2b-1;andthediureticactivityofthis Malpighian tubule fluid secretion of Manse-CAP2b is mid-
fragment Manse-CAP2b[2-8] (EC =8nM) matches that of waybetweenthatofthetwoCAP2b/PVKpeptidesnativeto
50
Musdo-CAP2b-1 (EC =9nM). The increase in activity the housefly; and thus can serve as a relevant model
50
observed with the loss of pGlu1 likely arises from the fact sequence to determine a structure-activity relationship
thatitnowmorecloselymatchesthesequenceofthenative profile. The C-terminal fragment Manse-CAP2b[3-8] retains
peptide.RemovalofanotheraminoacidresiduefromtheN- asignificantportionoftheMalpighiantubulefluidsecretion
terminus,asinManse-CAP2b[3-8]leadstoalargedropofan activity of the parent peptide (Table 1). The active core,
order of magnitude from the parent Manduca CAP2b the minimum sequence required to retain significant
peptide.Removalsofathird(Manse-CAP2b[4-8])andfourth diureticactivityinthehousefly,isthereforetheC-terminal
(Manse-CAP2b[5-8])residueleadtoanalogsthatretainonly hexapeptide. However, full activity requires theC-terminal
traceactivity.InthecaseoftheAlascanseries,replacement heptapeptide fragment (Manse-CAP2b[2-8], which is more
of the Arg7 (Manse-CAP2b[Ala7]) led to an analog with no active than the parent Manse-CAP2b and equipotent
significant activity, indicating that Arg7 is therefore the with the native Musdo-CAP2b-1 (Table 1). The results of
most critical of residues. Replacement of Val8 (Manse- the Ala-replacement series identify two residues, Arg7 and
CAP2b[Ala8]; EC =3500nM) led to a large drop in activity Val8, as critical for diuretic activity. The residue Leu2 is
50
(70-fold) in comparison with the parent peptide, and semicritical; whereas all others are not critical. Not
demonstrated retention of only 45% of the maximal surprisingly, the critical (R7 and V8) and semicritical (L2)
responseofManse-CAP2bandthenativehouseflypeptides. residuesareamongthemostconservedamongtheCAP2b/
Residues pGlu1 (Manse-CAP2b[Ala1]; EC =23nM), Tyr3 PVK family throughout arthropods. The most conserved
50
(Manse-CAP2b[Ala3]; EC =33nM), Phe5 (Manse-CAP2- residues among the CAP2b/PVKs are the C-terminal PRVa-
50
b[Ala5]; EC =115nM), and Pro6 (Manse-CAP2b[Ala6]; mide as well as a Leu at position 7 (equivalent to Leu2 in
50
EC =89nM) would appear not to be critical for housefly Manse-CAP2b) from the C-terminus [27]. The structure-
50
diureticactivityastheresultinganalogswereeitheralittle activityrelationshipprofiledeterminedinthisstudycanaid
more or a little less active. While Manse-CAP2b[Ala5] in the design and development of peptidomimetic agonist/
demonstrated a maximal response of 70%, this value is antagonist analogs of this diuretic peptide family with
not statistically different from that of the parent peptide. enhanced biostability and bioavailability as tools for
Leu2 would appear to be a semicritical residue, as Manse- arthropod endocrinologists and as potential pest manage-
CAP2b[Ala2](EC =373nM),provedtobeaboutsix-foldless ment agents capable of disrupting the water balance in
50
active. target insects.
60 peptides 28 (2007) 57–61
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