PWPBAN

Altstein Aliza

the and

R&D

chemicals.

ogano-chemical cxisting studics focused

inscct andlor protluct

Altstein(@l)

vinnie2@agri.gov.il; alizav@inter.net.il

CenterBet 510108.2008

lshaaya Horowitz (eds.). Biorotiorrtrl Corrtrr)I ofAr11rrr)pod DO1 16-2-3,0 Springer

1

The Rational Design of Insect Control Agents:

Family as a Study Case*

Miriam and Hariton

Introduction

The success of modern agriculture in developing and maintaining high-yield crops depends strongly on controlling insect pests by means of heavy use of insecticides, and at present organo-synthetic chemical insecticides remain the main weapon in this armory. However, in recent decades, uncontrolled application of chemical insecticides has led to acquisition of resistance by insects, has contaminated environment with toxic residues that endanger humans and other life forms, tlisrupted the ecological balance in and around cultivated fields. The growing concern regartling the toxic effects of insecticides has led to the implementation of strict regulations in the Western World, and these are being adopted by other countries too. These regulations limit the application of some organo-chemical insecticides and ban continued use of the more toxic ones.

The strategic approach that characterizes worldwide efforts is based on identification and development of novel families of non-toxic, insect-specific com-pounds that eventually could replace the organo-synthetic In seeking a new class of non-toxic insecticides that eventually could replace the existing toxic

compounds and overcome the limitations associated with the

has

bio-insecticides, entomological are on the search for targets and compounds that could form a basis for the development of highly effective, selective and environmentally friendly control agents insecticides, and emerge as a new mainstream of the insecticide industry.

M. and A. Hariton Department of Entomology. The Volcani Center. Bet Dagan. 50250, Israel e-mail:

"Contribution from the Agricultural Research Organization. the Volcani Dagan. Israel. No. series'

I . and A.R. Pests. 49 l0.10071978-90-48 1-23 Science+ Business Media H.V.2009

1.1 Illsect Netiropeptides Corttrol Agents/Insecticides

Inscct ncuropeptitles are prilne targets search novel insecticitlcs. regulatc Inany physiologicnl nntl tluring dcvclopn~cnt.

reproduction ancl (allt;~goliists) tlislupt alld intcrli.rc the IIOI.II~;I~ ;uid can yicltl. tI~creI'Orc, recel)tor-scloctive. insect-specilic insccticitles. Such

tlorivctl florn ant1 reselnble the natur;ll peptitles. hut woultl he ol' pcptitlonii~nctic chel~~icol iiature Nps them used

the hnsis design gcneric ol' i~isecticitlcs has nppliecl h11111an its ;I

the tlr~lg intlustry. Thc tnaior oyanislns, and li)r havc stilnulated

iuid those

Zit Nps: Per-spective

Since early IS)XOs, hecn idcntitietl, the hasic thcir (e.g., target cell. tlegradation) been physiolL)gy

have detcrmincd means setlucncing. receptor ill1

:~pplied association electrophysiological ant1 scc (Altstcin Niissel Giide Huuscr

et al. 'Taghert 2001; Nasscl ant1 Hornberg lirst the peno~nic/proteo~nic thc

tcchniclues and Etlman ant1 pt~tative tcstcd vitro

applied mainly identi Latcr. sequences were pretlictcd thc basis studies wcre carricd using ilnlnunohistochernicnl situ tcchniqucs liom membranes wcre sub,jccted specific binding These stutlics Icd discovery ofa fcw of less receptors bcli)re the latc

the fcw ycilrs. several atlvanccs I-cscalch Nps have enhilnced fi~miliarization their of thc imporli~nl IIicse ~ I ~ V ~ I I ~ C S WiIS thc sequencing several gcnonlcs ( I )mvol) l~i ln nlclarro,qcater.. Apis ~rrellifi.r.ri, Horrll>y.r nzori. Atloplrcl(~.r g~,trrril~i(rr and Ap(le.s rrc.h.yl~ti). yielded inforlriiition gcncs, on the basis rnany new 30-40 encotling

havc hccli prctlictctl each over havc been charactcrizctl. wcre the l'ly

11. rrielnrro,ycrster~, (c.p.. Lrrrcol~l~trerr nlci(l(~rae. Per.il>lorretrr N I I~C I .~~ -N I )O ) . (e.g.. L2oor.rto rrrigr.crtor.i~r ilnrl Sclri.stoc.c~rrcr grqaricr), v~irious

1.2

as

(Nps) in the for since they behavioral processes

senescence of insccts. Their blockers may with growth. development hehavior of insects and

antagonists would he have to a nature. The of enables to he as for the of a group insect-spccilic and non-toxic

- an approach that been to Nps novel direction in roles Nps play in the physiology of their high potential practical applications, active interest in Np studics in general. in of insccts in particular.

sect Historic

the many inscct Nps have principles of action biosynthesis, processing. release, transport, activation of the end have discovered, and their roles in the of

organisms hccn by of genome peptidomics, gene micro-arrays, characterization, and targeted gene interference.

in with physiological, behavioral analyses. For review and 2007; and Marco 2006:

2006: Hewcs and 2006). The insect Nps were identified (prior to era) by

classical of isolation degradation sequencing, their functions were in a variety of in vivo and in bioassays. This

approach was to functionally tied Nps. Np primary on ofthcir cloned pcncs. In parallel, localization

out and in hybridization and Np receptors, obtained fractionated cell

to assays. to the tens Nps and than live 1990. In past major in on inscct

our with structures and functions. One most of of inscct

which about Np precursor of which Nps were idcntilied. About genes

Np precursors in species. and a total of 150 inscct Nps most of which isolated from fruit

cockroaches locusts kincls of

Rational 5 1

(e.g., Maridrica sexta, gnmbiae Ae. aegypti).

i

ma.jority, peptide encodcd rtielarlogclster

peptide Peptides identitied, 11, ntelar~ogaster

mellifera. extended

method fentomole less)

desorption/ionization (ESI)

new

mode insecticiddinsect

issues (Altstcin Nbsel2007; Loof Gade Hauser and el al. Nassel and

Homberg Predcl al. 20117; Southey el 2008; 2000).

ant1 os agent made

thc pest-

and into agent. made; the bioavailabil-

peptides hintler

have tlevelopmcnt based

Design of Insect Control Agents

moths B. mori and various Heliothinae species), and from mosquitoes (An. and The great majority of these Nps were nsect-specific.

Genome sequencing also accelerated identification of Np G-protein coupled receptors (GPCRs). Within a few years 48 Np receptor genes, which constitute the vast perhaps all, of the GPCRs by the D. gcnome have been predicted and, to date, nearly 30 GPCRs in this fly have been characterized with respect to their preferred ligands. from other insects have also been and knowledge related to GPCRs was used to annotate 35 Np GPCRs in the recently sequenced gcnome of the honey bee A. The genomic information also our knowledge regarding Np prohormone-processing enzymes and several other genes that encode proteins involved in the regulation of cell-specific Np expression. This knowledge greatly extended our potential for gaining better and deeper insight into Np functions in insects. More recently, mass spectrometry (MS) followed by sequence determina-tion was employed for Np identification and localization. This enabled analysis of Nps (in quantities or in the hemolymph, in tissue extracts and single cells by matrix-associated laser (MALDI) MS, and examination of neuronal homogenates by electrospray ionization MS. A variety of MS-based approaches (direct tissue MS or MS in combination with laser capture micro-dissection) have accelerated the development of rapid and accurate identification of predicted Nps in small amounts of tissues, and even in single neurons, and have led to both identification and localization of hundreds of

Nps in a single step. This novel information, together with that obtained by means of more traditional

approaches, opened many opportunities for the use of "rational design" approaches to the discovery of receptor-selective Np agonists and antagonists. The resulting dis-coveries can further advance our understanding of the of action of insect Nps and may serve as potential management agents. Many recently published, detailed, well documented reviews of insect Nps address all the above

and De 2008; and Marco 2006; et al. 2006; Hewes Taghert 2001; Hummon al. 2006; Li et 2008;

2006; et al. Veenstra Although a vast amount of knowledge on insect Nps and their receptors has been

accumulated in recent years, in spite of the great potential of Np antagonists insecticides, not a single Np-based antagonist insect control has been commercially available up to now, and application of Np antagonists as control compounds does not exist. There are many reasons for this. First and foremost, no methodology has yet emerged for the conversion of an agonist to an antagonist

then, an insecticide or insect-control Second, even when such antagonists have been poor metabolic stability and the low ity of these - which stems partly from their difficulty in crossing mem-brane barriers and, especially, the insect cuticle - would their practical application as insect-control agents.

The very same problems severely limited the of therapeutic drugs on human Nps, therefore, recent studies in medicinal chemistry have

;limeti devclop stratepics overconit: stutlies inipressive propress tlcsipn. modilication. nntl

cherniciils. ant1 have Icd tlevelopment strntcgics for modifietl peptidcs ant1 ~nimeticagonistsantl;~ntagonists retlucctl

protcolysis and etlhanccrl bioavi~ilability. have improvetl chanccs ol'ohtaining drugs thitt s~ r i~c~ura l ly rcliitcd parent tuitl lei1 the tlevclopmen~ peptide-biised sevcral humi~ti tliseascs (Hiikfelt el al.

has been tnosr non-peptide ligantls fount1 rantlom of Iaree chemiciil

chen~ically i~ t l ap t~d impart sclectivc phiirnincokinetic Thcrc the

ngonist nntagonist, are being generation ant1 the niotlification ol'nativc peptides creak

bioovoi lithlc peptitlomirnctic colnpounils desirctl fcaturcs. tlrugs (Atlessi Soto

Hiikli.11 et al. 2003). few years.

lnsccl basetl ctcsign ovcrcolnes ol' ~ h c i~ssociated ;IS

paves towt~rtl devclopmenl mcthod applietl (PK)/pheromone activnting neuropeptitle

(PBAN) fitniily. sumtniwize thc ol'the PKIPBAN ofpcptides, untl dcscrihe iniplemcn-

tation of bioavailable

Tlte ZNA Appronclt

Thc itpproach

aponist an 9,.

an agonist idcntificntion (1 lead Thc l i rs~ steps towurds colnprise:

that the active agonistic Modilication nctivc serlttencc itlcntified

to to these problems. These have yielded in rational synthesis of

to the of several producing with susceptibility

to These new approaches, the useful arc to the

pcptitles, have to o f a few drugs for 2003) . Notwittistantling this progress the

technology not yet optimized and of the for Np receptors were by screening libraries and were then to the desired and properties. is still very limited information available on conversion of an to an and new approaches still sought, to the

of such antagonists to to stable and with the For detailed reviews on conversion of Nps to see and 2002:

In the past we have devised a novel integrated approach, designated Np Antagonist Insecticide, INAI - o n rational - which

some limitations with the application of Nps insect control agents nntl the way the of Np antagonists. The was to the pyrokinin biosynthesis

In this chapter we the INAI approach. review current knowledge family the

of this approach to the generation highly potent, selective and highly PWPBAN antagonists.

2 INAI Approach to the Development of Novel Insect Np-Based Antagonist Insecticides

INAI comprises two main steps:

I . Conversion of an into antagonist: Conversion of the antagonist into an insect-control agent prototype.

Conversion of into an antagonist requires of antagonist. this

I . Itlcntilication of the minimal sequence constitutes site o f the Np.

2. ol' the in step I.

2.1 I

Rn~ional Insect

3. peptides

peptides

agonists

al. al. al. al.

Maretro al. al. 198 1; al. al. 1 et :\I. Rose11 al.

Vavrek peptides

lo

tcsted Once

peptides sufticient

flexibility

peptides al. 1995).

or

peptide

peptide

pept ide(s) molecule(s).

molecule(s)

53 Design o f Control Agents

Determination of the bioactivity and structure-activity relationship (SAR) of the that are generated in step 2.

3. Identitication of a linear lead antagonist (among the modified examined in step 3).

The rationale behind these steps derives partly from practices developed in the field of medicinal chcmistry, in attempting to convert vertebrate Np to antago-nists. Many vertebrate Np antagonists were discovered by simple modifications of their primary sequences (Cody et al. 1995; Collins et 1996; Coy et 1989; Folkers et 1984; Heinzerian et al. 1987; Hruby et al. 1990, 1992; Llinares et 1999; et 1998; Piercey et Rees et 1974; Rhaleb et 199 : Rodriguez 1987; et al. 1983; Sawyer et al. 1981; Vale et 1972;

and Stewart 1985). In order to minimize the number of possible combina-tions to be examined and. therefore, the number of that require subsequent bioactivity evaluation. it is necessary identify the shortest possible active sequence in the native Np. Once that is known, sequential modifications (mostly based on substitution by D-Phe or D-Trp) are made to it, and the resulting small linear libraries are for bioactivity.

a linear lead antagonist has been made available it is necessary to improve its characteristics and to optimize it in accordance with its intended applications. In the present case, this involves generating a more efficient, highly potent antagonist that is metabolically stable, and that exhibits receptor-selectivity and bioavailability. Linelar cannot serve such a purpose because they are highly susceptible to proteolytic degradation, they lack bioavailability, and their high conformational leads to lack of selectivity. An effective approach to overcoming these limitations is through the introduction of conformational constraint into the linear lead peptides, which leads to higher resistance to proteolytic degradation, slower equilibrium rate and reduces the flexibility of the molecule.

Conformational constraint can be imposed on by various means (for reviews see (Hruby et 1990: Giannis 1993; Goodman of which cyclization is one the commonest and most attractive (Kessler et al. 1986). Conformational constraint: ( i ) imparts high selectivity by restricting the conformational space of the

to a conformation that mediates one function and excluding those that mediate other functions; (ii) enhances metabolic stability by excluding conformations that are recognized by degrading enzymes and thereby preventing enzymatic dcgratlation; (iii) increases biological activity, because of the much slower equilibrium between the conformations; and (iv) improves bioavailability by reducing polarity. However, these advantages are gained only when the conformational space of the cyclic overlaps the bioactive conformation.

Following the above steps, generation of an optimized antagonist involves:

5. Conversion of the linear to conformationally constrained 6. Determination of the bioactivity, SAR, selectivity, stability and bioavailability of

the obtained in step 5.

lrquirctl

itlentificd. developed agent. ~ntroduction

i.c.: anci ret~dily penetrate the

andlor conipounds environniental anti, Inust protluction. generation

cliaractcristics peptide (SM),

Biophorcscan detailctl co~nputational (agonistic ant1

magnetic crystallogrilphy) (Gilon 1998b; et et

be the pharmacokinctic thc peptidcs

cloncd thot be

the the non- peptide

hcen identified, generated stage

ol'the confonnational recluirements pcptides

Deter~nination nativc of nnd mode the the conformationally conslraincd

the the gathcretl steps

I nun-peptidic combinatorial librilries slcp the Ihr

ldcntification non-peptidic SMs.

non-pcptidic SMs have bioavnil- and be The hioavailablc

then sub.jected i~ppropriate licld

been some vcrtcblatc scc (Adessi Soto Hiikfclt al. 2003),

filr, needs tlevclopcd

7. Selection of a conformationally constrained antagonist with the properties (on the basis of step 6 . )

Once an improved antagonist has been i t must be into a prototype control This requires the of features that arc normally found in commercial insecticides. the antagonist must bc converted Into a low-molecular-weiplit compound, able to through inscct cuticle gut; the resulting must have high stability:

especially, be cost effective in The of small, cost-effective molecules with the above necessitates conversion of the antagonist into a non-peptidc small molecule and this, in turn, requires identification of the biophores that are essential for the antagonistic activity, and their incorporation into novel, scaffold SM libraries.

be identified through con formational and analysis of the active or antagonistic) inactive compounds (by means of nuclear resonance, NMR and X-Ray et al. Grdadolnik al. 1994; Kasher ct al. 1999; Saulitis al. 1992). Further SAR information can obtained by examination of relationships of the native and conformationally constrained with native and mutated Np receptors. This requires availability of both a receptor that can be mutated, and of a functional binding assay can developed into a high-throughput assay (HTA) for fast screening of

small-molecule (SM) libraries and selection of the biologically active compounds. Once biophorcs have SM libraries can be and screened for insect control agent prototypes. This final involves the following steps:

8. Determination antagonistic of the identified in step 7.

9. of the SAR of the and the mutated Np receptor, the of its interaction with native and

molecules. 10. Design of non-peptide SM libraries on basis of information in

8 and 9. I . Synthesis of SM on the basis of 10.

12. High throughput screening (HTS) of resulting SM libraries bioactivity. 13. of bioactivc

Once such been obtained. their in vivo bioactivity, ability stability must re-evaluated. most potent, stable and of them can be to formulation and preliminary toxicology evaluation and to testing in preliminary trials. Although this strategic approach has used in the development of Np agonists and antagonist-based drugs (for reviews and 2002; et i t has not. so

been used in relation to inscct pest control, and still to he in this context.

Insect 55

1 vivo

13

10

constrained

stcp depends.

PKIPBAN confornationally bioavailable

PKIPBAN

Identificationof Peptides

ant1 (Hclicovcrpu zea).

al. characterized H, Hez-PBAN sequencc

other scvcral cDNA;

(Altstein

(e.g., gcimbialc aegypti). peptidcs cloned were genome (Holt et al.

al. al.

Rational Design of Control Agents

An overall multidisciplinary strategy is necessary in designing an insect control agent prototype based on the INAI approach, and its success depends on the fulfill-ment of several preliminary requirements:

Knowledge of the primary sequence of the target Np, on which step depends. Availability of in or in vitro bioassays for screening the libraries and for selection of the most potent compounds, on which steps 3,6 and depend. Knowledge and know-how in molecular design and combinatorial chemistry, on which steps 5 and depend. Availability of or access to an advanced chemistry facility for synthesis of linear pcptides and of conformationally combinatorial and SM combinatorial libraries, on which steps 2, 5 and I I depend. Availability of or access to specialized facilities for determination of the molecular structural conformations on which stcp 8 depends. Availability of cloned native and mutated receptors that are required for steps 9 and 12. Availability of a receptor-based HTS assays (HTSA) for fast screening of SM libraries and selection of the biologically active compounds, on which 13

We applied the above strategy to the family of insect Nps, and thereby identified several highly potent constrained, antagonists, both selective and non-selective. There follows a short summary of the PWPBAN family and the steps that led to the identification of these antagonists.

2.2 PBAN and Family

2.2.1 Isolation and PBAN and Other Pheromonotropic

PBAN was first reported by Raina Klun (Raina and Klun 1984) as the Np that regulates sex pheromone production in female moths In 1989 Raina et al. (Raina et 1989) isolated and PBAN from zea, as a 33-amino-acid, C-terminally amidated Np, which was designated (for nomenclature see (Raina and Gade 1988)). Since 1989 the primary of 19 PBAN molecules has been determined in many moth species, by methods: sequencing of the purified Np; from cloned and from gene sequence or genomic data (for review see 2004) and Table I). PBAN molecules were also found in insects belonging to orders other than Lcpidoptera

in A. and in A. Those were not but predicted from the sequences of these insects al. 2002; Nene et 2007). Comparison among the primary sequences of the above molecules revealed that the pcptides share differing degrees of homology ((Jing et 2007; Wei et

56 iM. t\ltsrcin ,.\. Hiuiton

2008). Table ). of Iengtl~ and degree thern share cornlaon C-tern~inal pcntnpeptide FSPRLa.

Further hl~sctl ~nolcculur revealed sequence the cDNA encotling the prc-prohonnone Ihur

ant1 that separatetl prohormone ol'the hasic aniino Alg. Gly, Sor amida-

and that l i~ur pcptides have scrluuncc FXPRLn G tliapouse (DH). subcsophngcal g~ulglion P and one FXPKLil pcpritle (a-SGNP) (see Table /-Is the number scclucnccs incrensetl, the scqttencc h;w been extended include H. mor.i ant1 R. rrrtintltrr.irrtr-B- SCiNP) ant1 I). rrr(~ltrrro~qtr.ster scc T~lblc ). rcvcalctl ntltlitional vnrintions iis occurrence o f a kionctivc

homolog, ( i n /l.rcoti.s .selcrrtrr.i(r crrttrcccr) thn~ was Ibund he conncctctl lo

b-SGNP (Kawni el al. The gcne that encodes thcse live pcp~itlcs tcrtncd the gcne (Sato ct nl. et nl. and was found cxprcsscd seven ncurosecretory cells. termed the DH-PRAN-producingluci~ig ncurosccrcrory (DHPGs) the s~bcsophi~gcal g:unglion (SOG) (Si~to ct nl. 199.3. 1994). Examples the peptitlcs from i1 I'cw tnoth species listctl Tahlc

slutlies rcvcalctl other originate lioln thc the same sequence.

the PKs and Lom-PK-11); the tnyotropins (Lorn-MT-1 arc ~nyotropic isolated from the rriotlor.rrc~ i~litl

the 1,. rnigr-citor-irr (Nachmnn H~l t l l i~n Nachrnon el al. 1986: School> et al. I99 I993b): iuld ~nyolropic peptitlc S. .g~'t:qtrr.ia

l (VccliIcrt ct ill. 1997). Atltlitioni~l to shi~rc same scqucncc i\W: plncromono~ropin (Pss-PT) 18-amino-ocitl pcptitlc isolntctl liom I-'serrcIrrl(~titr (Mytlrirrrr~ci) sclptrrata (Matsu~noto ct ill. shares

P-SGNP: from I). rri~lrirro~qti.s/er- ( Kciui et al. iuntl (Meng et nl. of encotlcd :I

tlil'fcrcnt gene (Olscn ct nl. peptidcs li.om u~rr~r.iccrrrcr Pea-PK-5 Pea-PK-6 (Prcdcl oncl Predcl ct al. 1999). tlctilils oI' the amino acitl sctlllcnccs of some thcse peptitlcs see

nbovc scqucnccs were grouped one fiunily, rlesignatctl thc FXPR(K)l,n Ihlnily the PKIPBAN f'iunily, nanicd after ant1 Lcnl-PK

wiis the lirst ~ne~nbcr Si~mily he identilicd. (Holman ct al. and myosti~nulatory action ant1 pGlu at N-tcminus, (ant1 tcrmcd Recently, fcw ncltlitional the PRLa

C-lcrniinal sctlucncc were udclcd the fllmily; listed 'ri~hlc li)r li~rtlicr inf'orn~ntion thcir scclucnccs the reader referred Ral'L~cli nntl Jurcnka (Rafacli ant1 Jurenka

Thc tliscovcry oIaPBAN other stimulatctl Inany stuclics idcntilicntion iuid cl~li~ntilication new molcculcs, thcir gctic cxpl-cssion

and various untlcr vi~rious conclitions, tluring

and

and I Regardless their of homology all of a sequence

studies on cloning have that the of PBAN also encodes other pcptides,

in addition to PBAN, they are hy processing sequences. (single or pairs acids Lys or and lion) they are cleaved into which the PBAN conscnsus

(X = S. T. or V): hormone, two Nps (SGNP y), and I ) .

of available has the X in consensus to X = I (as in

K (as in PK-2, I Rcccnt studies in this family of pcptitlcs, such the

PBAN to hy a GR scqucncc 2007). was DH-PBAN 1993; Xu 1995). i t

to he only in pairs of' cells in

of DH-PBAN arc in I.

Advances in insect Np that pcptidcs. which do not DH-PBAN prohormone. also have signature They

include (Lcm-PK. Lorn-PK-I to IV). which pcptitlcs cockroach L.

migratory locust. and 1991; I, a from

(Scg-MT- ) pcptidcs that were Sound the consensus an

1992) that high homology with pcptitlcs - Drm-PK- I

2002) Drrn-PK-2 2002) - each which is by 2007): and two PK I? -

and - Eckert 2000: For of Table I . All pcptidcs

sharing the into which was or PBAN

which of this to 1086). had ("kinin"-like) a residue its thus pyro-kinin). a pcptidcs sharing just

to they arc not in I and on origins and is to

2003). and the PWPBAN pcptitlcs

o n isolation. of localization in insects their expression

TabIe other peptides PWPHAN fanlily - ," name 2 2.

Helicoverpn RQDPEQIDSRTKYFSPR1,a al. E

born by.^ LSEDMPATPADQEMYQPDPEEMESRTRYFSPRLa et al. p Born-PBAN-I1 Bornbjx rtiori RLSEDMPATPADQEMYQPDPEEMESRTRYFSPRCa (Kitanlura et al. 'n.

Brna-PBAN Bornby..r rnarzdnrirrn FSPRLa al. 2. Lyd-PBAN Lymnrzrria dispnr RPEPEQIDSRNKY FSPRLa et al. - 2 Has-PBAN He1ico1-erpn nssulra LSDDMPATPADQEMYRQDPEQIDSRTKY FSPRLa al. c. r, -

Agmris ipsilorr FSPR1,a (Duponets et 1999) 0 0

Mnrnesrra brassicne LADDMPATPADQEMYKPDPEQIDSRTKYFSPRLa Burnet 2 Spl-PBAN Spodoprern 1irrorclli.r LADDMPATPADQELYRPDPDQIDSRTKYFSPRLa (Iglesias C - PLx-PBAN Plr~relln xylosrella FSPRLa c

Helicoverpn nnnigern SDDMPATPADQEMYRQDPEQlDSRTKYFSPRLa al. 2004~) 2 Hev-PBAN Heliothis virescens ADDMPATPADQEMYRQDPEQIDSRRTKY FSPR1,a

Spodoprern esigitn FSPRLa al. Arip-PBAN Arirhemen pernji LSDDMPATPKDQEMYHQDPEQVDTRTRYFSPRLa al.

Sarnia cyrtlhia ricir~i LTEDMPATPTDQEMFDQDPEQIDTRTRY FSPR1.a al. 2004) Adoxopl~yes QSEAVTSSDEQVYRQDMSPVDGRLKYFSPRLa (Choi ec al. Mnruiuca sexfa ISEDMPATPSDQEYPMYHPDPEQIDTRTRYFSPRLa Denlinger 200.1)

Assc-PBAN Ascoris selerzar-in o-erncen QLVDDVPQRQQlEEDRLGSRTRFFSPR1.a al. Cln-PBAN Closrern ar~nsrorrrosis FSPRI-a al.

Orgjin rl~,vellirin LSDDMPATPPDQEYYRPDPEQIDSRTKYFSPR1,a Botnl?\~.r TDMKDESDRGAHSERGALCFGPRLa (Imai al. Hclico~:erpn ren NDVKDGAASGAHSDRLGLWFGPRLij al. 1994) Helicovrrpn nssulrn NDVKDGAASGAHSDRLGLWFGPRLa ct al. Helicoverpa nntrigem NDVKDGAASGAHSDRLCLWFGPRLg ct al.

Agi-DH Agmtis ipsilorr NDVKDGGADRAHSDRGGMWFGPRLa (Duportets al. Spodoptera 1irrornli.r NEIKDGGSDRGAHSDRAGLWFGPRLg (Iglesias k~cusm migrotorin pEDSGDG WPQQPFVPRLa (Schoofs al.

Lorn-PK-[I hcrrsm rnigmtorin pESVPTFTPRLa (Schoofs et al. 1993a) .A

4

1 Amino acid sequence of PBAN and of the

Code Insect species Amino acid sequence Reference

Hez-PBAN Born-PBAN-I

zen mori

LSDDMPATPADQEMY

Agi-PBAN Mnb-PBAN

LSEDMPATPADQEIYQPDPEVMESRTRY LADDMPATMADQEVY

LADDTPATPADQEMYKPDPEQIDSRTKY

Hnr-PBAN L RLKDSGLAPPDEYRTPELLDARAQY

Spe-PBAN

Scr-PBAN Ado-PBAN Mns-PBAN

sp

LSDDMPATPADQELY RPDPDQIDSRTKY

Orr-PBAN Born-DH Hez-DH Has-DH Hnr-DH

rnori

LADDMPATPSDQEY YRQDPEQIDSKSNY

Spl-DH Lorn-PK-I

(Raina et 1989) (Kitamura 1989)

1989) 00

(Xu et 1999) (Masler 1994) (Choi et 1998)

al. (Jacquin-Joly and 1998)

and Marco 2002) (Lee and Boo 2005) 0:

3

(Zhang et (Xu and Denlinger 2003) (Xu et 2007) (Wei et 2008) (Wei et

2004) (Xu and (Kawai ct 2007) (Jing et 2007) Unpublished"

et 1991) (Ma et (Choi 1998) (Zhang 2005)

et 1999) and Marco 2002) et 1991)

(continued)

(continued)

Codc Invct Refcrencc 'A cc

Lorr-PK Lecrcophnen ~nnderne pETSJTPRLa (Holnian al. Pen-PK-5 Periploriern n~~rericnncr GGGGSGETSGMWFGPRLa (Predel et al. 1999)

Peril>lnrrrrrr n~iirricn~rn WFGPKLa (Predel Drrii-PK- Dm.rol>hilrr ~rrelnrio.qcrsrer TGPSASSGLWFGPR1.a et al. Drlrl-PK-Zt' Droso~philn ~ ~ i r l a ~ ~ o g n s t e r SVPFKPRLa (Choi et al. 2001 ) LJI~I-MT-I L~crrsm migmrr~rirr GAVPAAQFSPRI-a (Schook et al. 1090a) Lj~ri-MT-11 L~ccrsm ~nigrcrroricr EGDFTPRLa et al. 1990h) L~rn-b1T- L~crrsrn rniglnrorin RQQPFVPRI-a (Schoofs et al. I990h) L~in-MT-VI L~c~rsrn ~nigrnrorin RLHQNGMPFSPKLa al. 199Oh) Scg-MT- Sclrisrr~ercn grqnr in GA4PAAQFSPRLa (Veelaen et al. 1997) Pss- Pserrdnlerin sepnmra KLSYDDKVFENVEFTPRLa (Matsunioto ct al. Her-P-SGNP Helicorerpn ,-.en DDKSFENVEFTPRLa et al. 1994) Hns-P-SGNP Helicoverl~a nzscrlrn SLAYDDKSFENVEFTPRLa al.

P-SGNP Helita~vr/>n nrnrigc,ro DDKSFENVEFTPRLa et al. Agi-P-SGNP A S ~ I ~ S il>.~i/Oll SLSYEDKMFDNVEFTPRLa (Duportets et al. 1099) Mob-P-SGNP Mn~nesrm bms.iiccre SLAYDDKVFENVEFTPRLa Burnct SpI-P-SGNP Spt~doprero 1irrornli.r SLAYDDKVFENVEFTPRLa (Iglesias Marco B~JIII-P-SGNP Boin6y.v ~nori SVAKPQTHESLEFIPRLa (Kawano et al. He:-y-SGNP Helicoverl>n :en TMNFSPRLa el al. Hns-7-SGNP He1icoverl)cr nss~tlrn TMNFSPKLa et al. 1998)

:I-SGNP Helicarerl>n crr~iiigerrr TMNFSPRLa (Zhang et al. 2005) Agi-7-SGNP Agwris il>silorr TMNFSPRLa (Duportcta et al. s Wrrb-y-SGNP Mnr~rrsrm br(rrsicc.re TMNFSPR1,a (Jacquin-Joly Burnet 1998) $ - SpI-y-SG P Sl~odo/>re,a lirromlis TMNFSPRLa (Iglesias 2002) S.

a BornL?\:r ~iiori TMSFSPRLa et al. 1

C Bold amino acid diapaust: n~elanization hormone: phcromone PT 1

u-SGNP same insccts (VIRPKLa). P- et al. - 2.

"GenBank F

'A the Ilu.

I

Table 1

name species Amino acid sequence

et 1986)

Pen-PK-6 SESEVPGM and Eckert 2000) (Choi 2001)

I I I (Schoofs

I (School's et

PT

Hnr-

SLAY

SLAY

(Ma (Choi et (Zhang

1992)

1998) 2005)

Hnr-

(Jacquin-Joly and 1998) and 2002)

1992) (Ma 1994) (Choi

1999) and

N and Marco Born-y-SGNP (Kawano 1992)

letters indicate conserved sequences. DH - hormone: MRCH - and reddish coloration PBAN - ?biosynthesis activating neuropeptide: - pheronionotropin: PK - pyrokinin: MT - niyotropin: SGNP- sub-esophageal ganglion neuropeptide. The sequence of is the in all Additional sequences of DH. u-. and y-SGNP can be found in (Jing 2007).

accession No. AB2.59122. member of the family but with difference that X = Lys or

a

Rational

(e.g., on enzy- niatic receptor(s)

2003). frame

al. al.

(e.g., Helicoverpa armigera) neu-

al. al. al. 2004a,

release

relationship; PKPBAN covered

2003),

PIUPBAN

peptides

al. al. nl. al. al. 2004b; al.

melaniiation al. i.c., al. et al.

i.e., (Raina

also

after FXPRIKLa

synthesize are C,,,-C,,

I1 C,,-C2,

linolenic linoleic via hemolymph

al. PKJPBAN peptides

main of

Design of Insect Control Agents 59

embryonic and post-embryonic development stages, bioactivity, modes of action cellular activity, second-messenger mediation, and their effects the pathway involved in sex pheromone production), and characterization of the

that mediate their functions. These studies are reviewed in (Rafaeli and Jurenka Phylogenetic trees, based 'on the open reading (ORF) sequences of known DH-PBAN genes were also constructed and were matched with taxonomic characteristics of the insects (Jing et 2007; Kawai et 2007). More advanced studies focus on gene organization, identification of transcription factors that activate the DH-PBAN gene in different moth species B. mori and

and the role of this activation in the differentiation of roendocrine cells (Hong et 2006; Shiomi et 2007; Zhang et 2005). Earlier studies, some of which are still being evaluated, addressed transport routes,

sites, target organs, and degradation of the peptides; these are reviewed in (Altstein 2004; Rafaeli and Jurenka 2003). In the present review we brietly sum-marize our studies and those of other workers on a few topics i n this field: (i) biological activity of the PKIPBAN family; (ii) structure-activity and (iii) characterization of the receptor. Additional topics were in three comprehensive recent reviews (Altstein 2004; Rafaeli 2002, 2005; Rafaeli and Jurenka to which the reader is referred for further information.

2.2.2 Biological Activity of the Family

The PWPBAN family of is a ubiquitous multifunctional family that plays a major physiological role in regulating a wide range of developmental processes in insects: pupariation (Nachman et 1997); diapause (Imai et 1991; Nachman ct 1993; Sun et 2005; Xu and Denlinger 2003; Zhang et Zhao et 2004); cuticular (Altstein et 1996; Matsumoto et al. 1990); feeding,

gut muscle contraction (Nachman et 1986; Schoofs 1991); and mating behavior, sex pheromone production and Klun 1984; Altstein 2004).

The first function that was discovered to be mediated by a member of the PW PBAN family - a function that provided the name of one of its peptides, PBAN - was stimulation of sex pheromone biosynthesis in female moths (Raina and Klun 1984). Now, nearly 25 years of research, it is well established that PBAN and other pcptides of the family regulate sex pheromone production in many moth species. PBAN stimulates sex pheromone production in insects that

type I pheromones (which composed of unsaturated primary alco-hols with a straight chain and their functional derivatives are produced from acetyl-CoA by de novo synthesis in the pheromone gland), and type pheromones (which arc composed of polyalkenes with a chain and their epoxy deriva-tives use and acids that originate in plants transported to the pheromone gland the after association with lipophorin, and only the epoxidation proceeds in the pheromone gland) (Kawai et 2007).

As studies on the bioactivity of the family of progressed, it became apparent that the roles this group of Nps are in development

1990s terminate

linal i.e., role FXPR/KLa

8. rnori, the peptides

induce next the targets tlevcloping

ol' (PG) later 5"' insti~r

inlluencc

peptides pupal their ecdysten>idogcnesis PG, caul-sc

(Zhang al. 2004b). tlirect e.g., factor, PG

DBIIACBP ecdysten)ids, result termination el al.

few Hclico~~erl~o/H~~liotI~i.~ virescetls Denlinger 2003). nrr~ii,r(cm al. 2004~)

H. nssultn (Zhao al. 2004). accelaated fleshfly (Sarroplzczgu Drillata). S. Olrllata

puparial

al. peptides

first Le~rccrnia separutu, al. hormone

niclanization (Ben-Aaiz al. et al.

at. I98 1 ; neurosccrelory gene pcptitlcs,

development holomctabolous rrzori

immature

and

et al. thc peptitle carried

e.g., stud-

processes, such as metamorphosis and diapause. Studies since the late have found that members of the family induce embryonic diapause. pupal diapause, accelerate pupariation and regulate the stage i n molting, cuticular melanization. Studies on the of the peptidcs have indicated that they induce embryonic diapause in through a mechanism that is yet not fully understood; i t could he that act o n the developing oocytes during pupal-adult development, and thereby diapause in the generation (Yarnashita 1996); or i t could he that hormone the ovaries, either directly, or indirectly through the DH receptors the prothoracic gland during the stages of the larva and the early pupal stages, thereby regulating production of ecdysteroid hormones that might post-embryonic development (Watanabe et al. 2007).

These are also involved in termination of diapause, through activation of in the in the of larval-pupal development

et Whether this activation is or indirect, via another is also unknown so far. Recent studies suggest that DH might stimulate the

by up-regulating expression of a gene and thereby causing an increase of which would in diapause (Liu 2005).

Termination of pupal diapause has been demonstrated in a species (H. (Xu and H. (Zhang ct and

et PWPBAN Nps also pupariation in wandering larvae of the Studies of revealed that Lem-PK accelerated the switch from wandering behavior to immobilization/ retraction behavior, and subsequently affected tanning. By accelerating both aspects of puparium formation, Lem-PK mimicked the effects of the pupariation factors, in exerting an effect on the central motor neurons (Nachman et 1997).

PWPBAN have been reported to be involved in cuticular melanization - the last step in the molting processes - in many noctuid moths. The possible involvement of this family of Nps in the control of larval cuticular melanization was observed in the common army worm, by Ogura and co-workers (Ogura 1975; Ogura and Saito 1972; Suzuki et 1976). The hormone, which was termed melanization and reddish coloration (MRCH), was later found to be identical with Bom-PBAN, which initiates the of the integument of many moth larvae, ct 2005, 2006; Hiruma 1984: Matsumoto et Morita et al. 1988).

PRAN cells, which express all live DH-PBAN were recently associated with another developmental function: maturation of the llight motor system of insects. Studies of B. revealed correlation between the rhythmic electrical activity of dorsal longitudinal flight muscles, on the one hand, and the bursting activity of PWPBAN neurosecretory cells, on the other hand, which suggested that the development maturation of flight muscles during pupal-adult development is regulated by PW PBAN peptidcs (Kamimoto 2006).

Most studies of various functions of this family were out moths, although a few were performed with other insects, gut contraction

and

ies tlies. stimi~lated

peptide peptides (Gade

al. al. number

peptides i.e., P

peptides

al.

vivo e.g.,

al. Gazit al. Holman al. 199 al. 1984; Schoot's

et I993b;

PKIPBAN

peptides

(FXPRLa; =

al. Nachman Holman 199 1 ; Nagasawa 1992).

lcss ct al. et al. al. 1992b).

Heliothis p~ltigeru peptides

12 full- and

(YFXPRLa) PBANI-33NH, analyzcd

post-in,jection al.

i.e., PKs, Pss-PT.

61 Rational Design of Insect Control Agents

in cockroaches and locusts; and pupariation in Studies performed in sev-eral laboratories, including ours, have shown that most functions can be by more than one member of this family, and that the are not species-specific; this subject is reviewed in 1997; Rafaeli 2002; Rafaeli and Jurenka 2003; Kamimoto et 2006; Ma et 1996). As the of DH-PBAN-sequenced genes has increased, particular attention recently has been focused on the multifunctionality of derived from the DH-PBAN-gene, DH, PBAN, a , and y-SGNP, as demonstrated in their pheromonotropic and diapause inducing (in embryos) and terminating (in pupae) activities. It has been found that

derived from the DH-PBAN gene, except for a-SGNP, can activate all pheromone biosynthesis and embryonic diapause; a-SGNP activates only on the former and not the latter (Ma et 1996; Sato et al. 1993).

The involvement of PWPBAN Nps in the above functions was demonstrated in a variety of in and in vitro bioassays, pheromonotropic, melanotropic, diapause, pupariation and myotropic assays, that were developed and optimized in several laboratories (Altstein et 1996; et 1990; et 1; Matsumoto et 1990; Nachman et al. 1993, 1997; Raina and Klun

al. 1991, Warek et al. 1998). All these assays, apart from the myotropic one, were carried out in vivo.

2.2.3 Structure Activity Relationship of the Family

Identification o f the amino acid sequences of PBAN and of other members of the PWPBAN family made possible detailed SAR studies that used synthetic derived from their sequences. Early studies on a variety of moth species have shown that the C-terminal region of the Np is essential for the pheromonotropic activity, and that within this region, the signature pentapeptide X S) represents the minimal sequence required for induction of pheromonotropic activity, although in most cases its activity was lower than that of full-length PBAN. The amide group and the X position were shown to be of major importance (Altstein et al. 1993, 1995, 1996, 1997; Kochansky et 1997; and et al. 1994; Raina and Kempe 1990, The N-terminal part of the molecule was much important for the onset of pheromonotropic activity (Altstein 1993; Raina and Kempe 1990; Kitamura 1989; Kuniyoshi ct A variety of Hez-PBAN-derived fragments in a range of doses applied to

at various times post-injection were used to demonstrate, that lacking or even 16 amino acids from their N-terminus were as active as the length PBAN, that a C-terminal-derived hexapeptide that contained the signature sequence was capable of stimulating a similar level of sex pheromone production to when its activity was alter shorter intervals (Altstein et 1995); this indicated that the hexapeptide might be the biologically active site of the Np.

Structure-function studies were also performed on other insect Nps that contain the PBAN pentapeptide C-terminal region, Bom-DH and All of these

pcptidcs sliowetl pheromonotropic nntl contirmed !he C-terminal onset (Ahern;~tliy et al. Foni~gy 1992; ct al. 1992a: Nachman et nl. 1993; Schoofs et al. 1993h).

evnluntion ant1 stutlies the PKI ant1 conli>rrnationally

hintctl that 0-turn constilutes the thcsc pep- lides. orcler provitle n dclinilive evidence p-turn actlve cxoct type p-turn ( I . 1' 11). (E)-alkcnc trr~rrsPn) isostcric recently bccn by four PKI

hioilssay systems. conformationally constrained PK-Etz demon- stratctl cquivalcnt parcnt peptidcs four

matchctl and/or :~ctivity peptitlcs them. Tlic relatively potent agonist ilctivity

evitlcnce t,nrl.rPro P-turn represcnls conformational

rilngc (Nachman et al. 2009d). dcscrip- tion rcsults Nachman

The pcptidcs stimulate sex raised tlic cluestion whctlicr diffcrcnt peptidcs tilight be metliated by rnultiple pcptides mecliate

iictivity by Tlic multi-func~ioni~lity ol' several different insect raisetl questions clifl'erent dil'ferent

locatctl tlill'crcnt tiirgct each niccliatctl satlie Those ant1

cluestions led PKIPBAN

hiochcinical, itnd histochctnical were used rcccp-

this lhrnily m:~,jority the stutlies focused mctliate phcromonotropic stutlics

that act target the (scc (Altstein hccalnc clear.

doubt, mediatetl the the pheromone

histochemical study (Altstcin al. the gland cells of /)ol/i,qeru the first clirect indicalion prescnce

the 'The out using hiotinyla~ccl photo- ilflinity (henLophenon ligands PBANI-33NH,

BpaPBAN I-33NH,, from C-terminus. Arg2'-PBAN28-33NH,, B ~ ~ P B A N ~ ~ - ~ ~ N H , ) , revealed

activity. the importance of region in its 1995: et al. Kuniyoshi

Further of PKIPBAN SAR conformational of' PBAN active core (based on NMR activity analysis of con-straint analogs) a active conformation of

In to more that the represents the conformation for the PWPRAN Np family and to try ancl identify the

of the or a PWPBAN analog PK-Etz, incorporating an component has evaluated us in diverse

PBAN The analog activity to PWPBAN of equal length in all PW

PBAN bioassays, and approached the of of natural length in three of of PK-Etz provides strong that a type I an important aspect of the PWPBAN Nps during their interaction with receptors associated with a

of disparate PKIPBAN bioassays A detailed of these is presented hy R.J. in another chapter in this book.

ability of a variety of to pheromone production the pheromonotropic activity of the PRAN receptors or receptor sub-types, or if all

this .

one receptor to which the C-terminal part of the PWPRAN Nps hinds. the PWPBAN family in species

similar as to whether the functions are mediated by receptors, on organs. activated by one or more of these Nps or whether all functions are by the receptor. other

to an extensive study on the receptors as indicated below.

2.2.4 PKIPBAN Target Organ and Receptors

A variety of techniques - molecular. physiological, pharmacological - to localize, isolate. clone and characterize the

tors of of Nps in various insects. The great of on the receptors that the activity. Early

hod suggested PBAN might on a other then pheromone gland 2004) and references therein), but since the late 1990s i t

beyond any that the pheromonotropic activity of PBAN is via PKIPBAN receptor in gland.

A et 2003) of pheromone H. was of the of the PWPBAN receptors

in pheromone gland; study was carried two substituted) PBAN (a full-length

molecule. and a shorter fragment derived its and the presence of the

lintionnt Dcsign Control Agcnts 03

intersegmental membrane bctween, the abdo~ninal untl

abtlominal segment. revealed stiiining the cells.

likely eflicient hemolyniph and hormones, e.g., sex

these (letailed histochclnical stutly Altstein et al. (Altstcin al.

[he case other vertcbriite iind cellular

transmcmbrane (7TM) sense ccll and activ;itc cellular

Mtiny insect inclutling of PKI f~itnily werc stimuliltcd thc I.ro.sophila Geno~rie

Tilghert 1, and Dro.sol~lrilcr genome iuid the sequence (Adems et Holt

than years the cDNA 12 PKIPBAN (mainly ~notlis) and

were assulnption Tiighert may

ncuromcdin U (NmU-R), since thcir NmU motifs

i.c.. FRPRNa FSPRLa, Primers based the NlnU CG8784 CG8795, CG99 CG

l'or Drvso/~hilcr Genome were succcssl'ully I) . ~rrel~r~logtrster

A1lol~1iele.s I). n~elcrrrogcr,ster- H, trzori H. Zen

(Table hiecd the sequences Sl~odogre,n littorc/lis crlmigcw,

Table atlult female li-om dill'crcnt instars

ccll Xewoprrs oocytes mot-i anti 1ittorcrli.s NIH3T.3

embryonic (HEK293). the B. mori

mori pupal gcnc strttclure,

tissue i~hility FXPRLa PRLa peptidcs. Phylogenetic trees werc constructctl

xceptors. Ibund be H. zeo al. 2003), H, vir.c.scens ct al.

2008) R. mori (Hi111 et al. Sound most thc

and, cases (Born-PBAN-R), nzori-R

of Insect

rcceptor in columnar epithelial cells throughout the eighth and the ninth segments, also in the ventral and

dorsal epithelial cells in t he ninth Staining a polar pattern, with intense at the basal part of epithelial This polarity of the PBAN rcceptor most facilitates contact with the

the blood-borne PBAN. that stimulate pheromone produc-tion in cells. A summary of the was presented hy et 2003).

As in of invertebrate Nps, tlie PKIPBAN Nps activate processes via G-protein-coupled rcccptors (GPCRs), which are seven domain proteins that molecules outside the

internal signal transduction pathways and, ultimately, responses. studies on Np GPCR cloning, those tlie PBAN by Project (Hcwes and

2001 by the completion of the sequence in 2000 of Anopheles genome in 2002 al. 2000; et al.

2002). In less 5 of receptors from six insects wcre cloncd another live were only annotated (Table 2). Most

of the receptors cloncd in light of the of Hcwes and (Hcwes and Taghert 2001) that the PWPBAN receptor (PWPBAN-R) be homologous to the mammalian rcceptor ligands - and the PWPBAN Nps - contain similar at their C-terminus,

and respectively. on consensus sequences of and on sequences of genes and 18 and 14575, which wcre annotated as coding GPCRs in the Project, wcre designed and used to clone two receptors (PK-2-I and PK-2-2), two receptors (PK- I and PK-2), PK-I receptor (PK-I-R), and also a and an PRAN receptor (PBAN-R) 2). A few receptors were cloned later on on already published PWPBAN-R (from and H.

2). Receptors wcre cloned from pheromone glands of moths, several larval and from pupal ovaries. Most rcccptors were

expressed in Chinese hamster ovary (CHO) or Sf-9 lines;.one was expressed in (R. pupal ovary), one (S. larvae) in and

hurnan kidney cell lines In all of the studies, other than pupal ovary, expression was monitored by Ca influx; in the case of the

ovary rcceptor i t was monitored by voltage clamping. The receptors were characterized with respect to their homology

B.

with other cloncd PKIPBAN rcccptors, distribution, and to bind or in searching for

orthologous All the cloned receptors were to GPCRs, and tlie pheromone gland rcccptors from (Choi et (Kim

and 2004) were to share a significant homology. However, the homology between the various PBAN-Rs deviated at N-terminus in some also in the C-terminus. H.

c J-

FXPRIKL-HN,

Tissuc GenBank Acc. #

Helico~,c,rl)n :en 19x52 B o ~ r r b y rnori AB181298 Helio111i.e vire.vcr~rs EU000527 CHO

(Choi et 2003) et al. 2004) et

1

Dro.co/~lrilo 1ne1n1ro~n.erc.r Dm>-PK- -R instar) (Cazzamali el al. 2005) Spodol~rern lirrornlis instar) Do407742 NIH3T.3. HEK2Y3 al.

7,008 Heliorlris ~.iresccrrs Hev-PBAN-R instar) EU000525 et al. Helior11i.c ~*ire.cce~u (B) instar) EU000526 CHO (Kim et al.

Pupae B o ~ n b y . ~ 111ori Bom-DH-R 161386 oocytes (Honilna et al. 3006)

Drosol~hiln ~rreln~logasrer Drosophiln ~irelnrrognsrer '277899 A11oplre1e.v gn~nbine A1rq~lrr1c.e gnrnbioe AY9002 CHO

A1rol~lre1c.c gnrnbine (PK-2) - Alrripheles gnrrrbine - Plrrrelln sylosrelln - H e l i c o ~ ~ e r l ~ n nnnigera -

(Robcnkilde et (Rosenkilde et al. 2003) (Olsen al. 2007) (Olszn al.

I er al. 2 - et al. .a -

E. z 2

Orgjio rhyelli~ln - AB28.3011 - ? HEK293: Human 1:

5. - 3 3

Table 2 Suniniary of cloned receptors

Insect Code Expression sys. Reference Pheromone gland

Hez-PBAN-K Boni-PBAN-R Hev-PBAN-R (C)

Pheromone gland Pheromone gland Pheromone gland

AY3 Sf-9 Sf-9 (Hull

(Kim

al.

al. 3008)

Larvae I Whole larvae (3rd AF36827.3 CHO

Spl-PBAN-R Whole larvae (5th Sf-9 (Zheng er 2007) Hariton. (unpublished)

(A) Larvae CNS (5th CHO (Kini 2008) Hev-PBAN-R Larvae CNS (5th 2008)

Developing ovary (pupa) AB Xenopus

Whole insect Drm-PK-2-R- I Whole insect AY277898 CHO al. 2003) Drm-PK-2-R-2 Whole insect AY CHO Ang-PK-I-R Adult whole insect AY9002 I 8 CHO et Ang-PK-2-K Adult whole insect 19 et 2007)

Annotated only APR-I (Rosenkilde 2002) APR-2 (PK-2) (Rosenkilde 2003) Plx-PBAN-R 2005 Har-PBAN-R 2005 aa

On-DH-R 2007

CHO: Chinese hamster ovary cells: embryonic kidney cells.

al. nanomolilr

peptides peptides

niori eclasion. mod

al. aFfinity peptides

was littoralis

(Zheng al. receptors,

(PKC)-dependent Gai-independent.

peptides melcrnogaster: Dr~n-PK- -R (Cazzamali

al. Drtn-PK-2 Dun-PK-2-1 Dnn-PK-2-2 al.

Drosophila CG9918, CG8784 CG8795, Drm-

peptides Drm-PK-2-1

Dnn-PK-2 (RNAi) whercas

first instar al. Ang-PK-2-R, (Olsen

al. peptides Ang-PK-2.

Alig-PK-2

PRLIINa (Capa-I, and FXPRlKLa

capa Dmsol~hilu

Further al. Olscn al. the

Plrrtella xylostella arnticqern) thyellirln

Rational Design of Insect Control Agents 65

contained an extra amino acid sequence on its C-terminus, which was found to be responsible for the regulation of clathrin-mediated internalization of this receptor after i t was challenged by PBAN during pheromone biosynthesis (Hull et 2004, 2005). All pheromone gland receptors responded to PBAN doses in the range, in a dose-dependent manner, and also to derived from the DH-PBAN gene products and to other FXPRLa and PRLa - but at lower affinities. The pheromone gland receptor of B. was found to be tissue-specific, and it under-went significant up-regulation on the day preceding Another B. recep-tor of the family was cloned from pupal ovaries during pupal-adult development (Homma et 2006). The receptor exhibited high toward DH doses in the nanomolar range. The affinity of the receptor to other DH-PBAN-derived

much lower. We have cloned another PBAN-R from fifth-instar larval tissue of S.

et 2007). 'The receptor also showed high homology with the pheromone gland with differences in the N-terminal region, the second outer loop and the third inner loop. PBAN induced activation of an MAP kinase via a signaling mechanism that was protein kinase C but As in the case of the pheromone gland receptors, the larval receptor also responded to other PWPBAN by MAP kinase activation.

Three PK receptors were cloned from D. I et 2005) and two receptors - and (Rosenkilde et 2003). These receptors are the first PK receptors predicted on the basis of the findings of the Genome Project, based on the homol-ogy with neuromedin U, and respectively. The PK-I-R was highly specific to Drm-PK-I; i t reacted with all other FXPRLa and PRLa but at much lower affinities. The other two receptors, and Drm-PK-2-2, exhibited high affinity, in dosages in the nanomolar range, toward only. Gene silencing by means of RNA-mediated interference

techniques showed that silencing of the PK-2-1 gene killed embryos, silencing of the PK-2-2 gene resulted in reduced viability of both embryos

and larvae (Rosenkilde et 2003). Two PK receptors. Ang-PK-1-R and were also cloned from the malaria mosquito A. gambiae et 2007). Similarly to the Dmsophila receptors, the Ang-PK-I-R was selec-tively activated by Ang-PK-I but not by The Ang-PK-2-R was less selective and could be activated by both and Ang-PK- I , but with a somewhat lower affinity for the latter.

A few other receptors that belong to the Np family Capa-2 Hug-y) and which share some homology with the family and are

encoded by a prohormone and PK prohormone genes (which also encode for PK- I and PK-2 in and Anopheles), have also been cloned and character-ized. information on their structure and characterization can be found in (Iversen et 2002; et 2007). Two other PBAN-Rs (from moth

and H. and one DH-R from Orgyia have been annotated (see Table 2).

nntl Hariton

Zmplernentatiorz ZNAZ PKIPBAN

Antago~iists

Initially andlor and pcptitles: phero-

pelt ige~n atlults. littornlis 1995,

peptides fronl Hcz- 1-33NH2, techniqiles identify

the PKIPBAN same

peptides phcrornonotro-

the -33NH,, that

SO%, (Altstcin

conformational

al. al. actlve PKPBAN family YFSPRLa), lead

confonnationally al. 1999a). sub-

secjuence (RYFSPRLa). lead All

peptides the a~nide

1). carriccl hid

et 199 1998a). revealed

led sex

-33NH, agonistic i~ctivity m scc I).

arnino 1999a; Zltser al. 2001). lnodified

21, peptides 3,

M. Altstein A.

of the Strategy2.3 for the Family

2.3.1 Discovery of PWPBAN

we optimized two in vivo bioassays for evaluation of agonistic antagonistic activities of linear conformationally constrained a monotropic bioassay with female H. and a melanotropic bioassay with S. larvae (Altstein et al. 1996). Once the assays were available we synthesized a variety of linear derived the sequence of PBAN and used SAR to the minimal active sequence of PBAN that constitutes active core of the molecule. The sequence that exhibited the activity as that of the full length PBAN was YFSPRLa (Altstein et al. 1993, 1995, 1997). A "biased library" of linear peptides, based on this hexapeptide active sequence, was synthesized, with each amino acid being sequentially substituted with the amino acid D-Phe. We tested the in the D-Phe substituted linear library for their agonistic and antagonistic pic activities by using full length PBAN, Hez-PBAN I as a stimulator, and discovered a highly potent antagonist - RYFdFPRLa - was capable of inhibiting sex pheromone biosynthesis by at a dosage of 100 pmol et al. 2000; Zeltser et al. 2000).

The discovery of a lead antagonist enabled us to take the next step: the search for improved selective and metabolically stable antagonists. For this we designed backbone cyclic (BBC) peptides, which exhibit constraint and offer many advantages compatible with the requirements of improved antagonists (Gilon et 1991, 1993; Kessler et 1986). The minimal sequence (that includes the consensus sequence of the and that of the antagonist (RYFdFPRLa) were used as a basis for the design of two constrained chemical BBC libraries (Altstein et The first, the Scr library, was based on a slight modification of the active The second, the D-Phe sub-library, was based on the sequence of the antagonist. the cyclic in each sub-library had the same primary sequence and same location of the ring; they differed in their bridge sizes and in the position of the bond along the bridge (Fig. This part of the study was out in collaboration with the laboratory of C. Gilon, of the Hebrew University of Jerusalem, who developed the BBC methodology and the cycloscan concept (Gilon al. 1, 1993,

Screening of the two sub-libraries for phcromonotropic antagonists that all the antagonistic peptidcs originated from the D-Phe sub-library, and to the discovery of four compounds that, at I nmol dosage, fully inhibited pheromone biosynthesis elicited by I pmol of PBANI and exhibited no (BBC 20, 22, 25 and 28; n + = 2 + 3; 3 + 2; 4 2; 6 + 2, respectively, Fig. Substitution of the D-Phe acid with a Ser resulted in a loss of antagonistic activity (Altstein et al. Four small BBC peptides, at their C-terminus (Fig. and four precyclic (Fig. based on two of the

et

Flg. peptides

(CH2),- -(CH2),

I

(CH2),- CO-NH -(CH2), peptides

(CH2),- -(CH2),

n=6;

peptides

(CH2)n

Peptide

(BBC20 BBC28;

peptide al. Zeltser al. peptides

Rational Design of Insect Control Agents

1 General structure of backbone A. Ser BBC sub-library cyclic (BBC)

CO-NH

I

B. D-Phe BBC sub-library

Fig 2 General structure of the "small" 28.1 RRC I I

CO-Arg-Tyr-Phe-D-Phe-Gly-Arg-NH2

28.3 CO-NH I I

CO-Arg-Tyr-Phe-D-Phe-Gly-NH2

m=2

Fig. 3 General structure of the precyclic NHY

I I

X Y n m

andBBC antagonists n + m = 2 + 3; 6 + 2. respectively), were also synthesized; their activities revealed that a negative charge at the N-terminus of the

eliminated the antagonistic activity (Altstein 2004; Ben-Aziz ct 2006; et 2001). Assessment of the metabolic stability of the BBC

indicated that they were much more stable than their linear parent molecules (Altstein

CI al. I909n. 1 ). Thcse compountls werc lirst :uitl scrverl ir lx~sis for design ol' other irnt;yonistic peptidcs Naclim:ui et ill.. 2 0 0 9 ~ ) of this Ih~nily.

tlie SAR, Uioavailal)ility tlie I3BC

'The av:lil;lhili~y a library conformation;~lIy rcccptor sclcctivityi whicl~ sonic peptides potcnt

nn~ngonisrs PBAN-cvoketl plicron~onorropic uctivily some ricvoitl of biologicnl ~lladc peptitles irlso \!cry powerhll

motlcs action ol' Ihc fi~tiiily cxhihits ~1 ~iiultifi~~ictionnl patlcrn ol'activity. enabling idcntilication selcctivc nntl non-sclccti\lc i~ntogonists li)r the irctivitics. ciln further provitlc inl'or-

wlicther the various l'i~nctions regulated by the tlil'ferent PKIPBAN mcdiatcd by [lie same rcccptor hy tliffcrcnt receptor subtypes. andl

more then one Such inli)rmntion Sol* l'urlhcr dcsign ol'

insect ;lgcnls t h a ~ 1nc:uit intcrfcrc with the lilrnily The high stirbility the BBC peptitics es their potentially

incrcescd hioavailability illso cnabled cvi~lui~tc thcir hioavailahility. and interl'cre endogenous. i.c.. ni~tivc

mcchanisn~ i~ction. The thcsc ex:uminirtions necessary hccnusc the hioassays thitt were used select the werc ol' n synthetic ant1 of may l'ully rcflccr the nnturnl (irr ~ ~ i ~ ~ o )

the pcptidcs inhihit sex phcro~none hiosyn- tlicsis elicitctl by i.c.. peptides, conlirmcd the tlitl the cntlogcnous nlcchanism. potcnt anti~gonistic itnd m 6

respcctivcly. scc ). werc able inhibit biosyntlicsis hy hX(;/c>, 57%. 54% 70f2.. li)r 11. I'ollowing in.icc~ion n~llol tlosagc (Altstcin cxi~~nination Ihe time ol'the potcnt

(BBC-28: n m h 3,. scc Fig. signilican~ inhihition 01' sex ~ ~ e l t i , q e ~ r / fcmalcs coultl I1 (Allstsin 2003), the potency ant1 nlctaholic s~;lhility an tag on is^. The

pcptitlss irlso tested their bioavnilability by thcni topically fcnlalc ~noths scotopliase ant1 thc 01' cntlopc-

nously clicitctl sex phcron~onc protluction. The iuitl BBC- m sce exliihitecl bioavailahility (i.c.

cuticuli~r pcnc~lxbility) ant1 were able pheromone protluction by 40-60% ond 50-67'2,. rcspcctivcly. siniilnrly lo thcir perlhrnlancc injcctctl tho inscct Hariton Altstcin. unpublislictl).

Furthcr evaluation of the bionctivity ol' the nntl ol' some ol' ~Iicir i.c.. s~nall peptides ant1 prccyclic pcptiiics, involvctl :I series ol'

200 the antagonists of the PWPBAN Nps as the (R.J.

2.3.2 Determination of Bioactivity, Selectivity, and of Antagonists

of of constrained BBC pcptitlcs (which confers high in

of and any activity the BBC tools for exam- ining tlie of PKIPBAN that

of potential various which important

motion on pcptitlcs arc or or Np. is indispensable

control are to activity of this of Nps. metabolic of well as

biological activity and to SAR. ability to with the

of last ol' wits to antagonists based on injection

stimulator elicitation an "artificial" response which not mechanism of action.

Evaluation of ability of the BBC to endogenous factors. by the natural that

BBC pcptitlcs inhibit Four highly BBC pcptidcs: BBC-20. 23. 25 28 ( n + = 2 + 3: 3 + 3: 3 + 2:

+ 2. Fig. I to sex pheromone nntl rcspcctivcly, 5 of a I

2003). Further of response most antagonist + = + I ) showed that

pheromone protluction in H. last up to I I indicating high of this

BBC wcrc for applying on at examining resulting inhibition

BBC pcptitlcs (BBC-25 28: n + = 4 + 2: 6 + 2. rcspcctivcly, Fig. I) high

wcrc wcrc

to inhibit when to

(A. and M. 2008 BBC pcptitlcs

analogs. BBC

Rational Design 69

that cxatiiined their PWPBAN-metliated melanization, hintlgut

Lom-MT-11.

different peptides peptide

peprides.

ditl'crcnt Ihc peptides

PBANI-33NH,, against melanotropic

more one compounds clici- PW

the itnd (Altstein al. 2 0 7 ; Ben-Aziz al. pcptide

the stimulated efi'cctivcly peptides

3, see mclanotropic Ixm- of

Ma,jor peptides melanotropic selective

(e.g., Ibund, Ihree BBC-

28- and In et Five melanotropic

were (BBC-20. m 3. I, scc 3).

m peptide pupariation cndoge:

nous affcct elicited in.jected be

the sclcc- phero-

non no tropic) pure agonistic Ben-Aniz

stitnulatory dilkrent the by

may result between thc e.g., tliffcrent possiblc that

rcccptors the pcptidcs d~ffercnccs

of Insect Control Agents

experiments ability to inhibit other func-tions - cuticular pupariation and contraction - elicited by PBAN and other member of the PWPBAN family such as PT, Lem-PK. Particular emphasis was placed on the question of whether elicitation of a given function by several and elicitation of several different functions by a given were affected by the same or by different BBC

Interestingly, the experiments revealed differing patterns of inhibition by the BBC pcptides when a given function was elicited by pcptides (Altstein et al. 2007). In pheromonotropic assay. BBC that inhibited pheromone biosynthesis elicited by did not exhibit antagonistic activity

any other tested elicitor. In the assay most BBC pcptides inhibitcd than elicitor but only a few of the inhibited all tors (Altstcin et al. 2007). The inhibition patterns differed among the other PBAN-mediated functions also, and in general there were marked differences between BBC inhibition patterns of the pheromonotropic and the pupariation

myotropic assays ct ct 2006). N o BBC inhibited myotropic activity, although this activity was by Lcm-PK, which was inhibited by two BBC - BBC-22 and BBC-23 (n + m = 3 + 2, and 3 + respectively, Fig. I ) - in the assay. PK-elicited pheromone biosynthesis was also not inhibited by any the BBC pcptides. differences were also found between the BBC that inhib-ited the pheromonotropic and activities. Four antagonists

exhibiting an inhibitory activity toward only one function) were of which were melanotropic selective antagonists (BBC-23, n + m = 3 + 3,

20-L- I, see Figs. I through 3) and one pheromonotropic selective inhibi-tor (BBC-22, n + = 3 + 2) (Altstcin al. 2007). non-selective and pheromonotropic antagonists found 25, 28, n + = 2 + 4 + 2. and 6 + 2, rcspcctivcly, 20-L- I and 28-L- Figs. I through only one of which (BBC-25, n + = 4 + 2) also inhibited pupariation. However, it is interesting to note that this inhibited when i t was elicited by the

1

mechanism, whereas i t did not it when it was by exogenously LPK. This indicates that the endogenous mechanism may not mediated

by LPK. Selective agonists were also found amongst BBC peptidcs. Six tive pure melanotropic agonists and one non-selective (mclanotropic and

compound were discovered (Altstein et al. 2007; et al. 2006).

The differing inhibitory and patterns that were found in assays indicated that various functions may be mediated structurally different receptors which do not equally recognize the BBC pcptides. Although the selectivity from differences assays themselves.

insects, developmental stages, assay conditions. ctc., i t is also it may indicate diversity in the binding pockets or in the ligand docking

regions of the elicitor on the that mediate the different functions resulting in an inability of BBC to block all of them. The obtained

givcn lnediate ligands niight

exhibit differing towartl receptor would. therefore, the

used stiltly the scqucnce FXPRLa the active the copohilirics elicitors givcn

assay also suggest (see

conipletion thc 01' ;1 large

and anti Thc tlnta Ibr fi~nction, the resitlues propcrtics penetrate

large gained recently (i) hioavailahility the I,PK MT) 2009a); the hioavailahility P'

P? substituted al. 2009b; Nachninn al. 2009n); (iii) ilnd tr.crrr.sPro Ac-YF[EtzlRLa and Ac-YF[Jo]RLa (Nachman et 2009b: Nachmiin nl. 2009d) (E)-alkene "Etz"

dihydroimitlnzoline tnoicty abbreviated "Jo", and n and linear

molecules. pcptidase peptidus cytotoxic Altstcin R.J. unpublished), set for

the Onc successful implel~icntation amphiphilic

Hex-Suc-AtlFPRLa the

pcptitle ofthe the PKIPBAN (FXPRLn) results

agonist (RYFSPIILa) (RYFdFPRLa) (Zeltser ct ill. The analogs Hex-Suc-AciFPRLa proven potent cfticacious ol' pheromone

(54% 100 The also Sound pure iIS f;~ilcd mclanization elicitcd ot'the peptides

transmigrate isoliitcd cuticle disscctcd atlult femalc Heliotlzis viwscc~rr.~ extent ol' 25-30f% ( 1 representing

(Nachmitn ct nl. 2 0 0 9 ~ ) . Results thc rhc D-Phc P-substitutctl analogs, PK-Etz. and PPK-Jo) detailed and su~nlnarizctl Nachman

nnother chapter

within a assay also suggested. that the PKIPBAN pcptides might thcir activity via different receptor subtypes. o r that the various

binding properties. with respect to docking or affinity, a given and differ in thcir degrees of inhibition by RBC pcptides. Given that all the elicitors in this contained same signature - - which constitutes site, differing inhibitory of the BBC pcptidcs towartl different in a

that thcir mode o f inhibition is non-competitive also item 2.3.3. below).

The of this stage of the study was marked by accumulation amount of information on the SAR of the BBC pcptides. on their

endogenous bioactivity bioavailability. on their selective properties. also identified the most potent antagonists each highlighted

that give the compounds their inhibitory and ability ro the cuticle. and shed important light on their non-competitive nature.

All of the above information together with the amount of data on: the high of native linear elicitors (PBAN. PT,

and (Hariton et al. (ii) SAR anti high of anti amino acid pcptides (Hariton et et

the bioactivity selectivity of the mimic analogs al. ct

(which contain an termed "Etzkorn" abbreviated moiety or a termed "Jones" respectively);

on the bioactivity of variety of amphiphilic hydrophobic and cyclic resistant and magic bullet analogs. (M. and Nachman, the basis further structural analysis in support of design of improved antagonists. example of a

of our approach is the design of the novel linear D-Phe analogs (in which a hexanoyl-(Hex) moiety linked by a succinic acid was incorporated to the N-terminus). The design of

was based on our finding that substitution second amino acid i n the consensus sequence of family hy D-Phc in conversion of an to an antagonist

2000). was to he a and inhibitor sex biosynthesis elicited by PBAN (84% at

100 pmol) and PT at pmol). but not by MT and LPK. analog was to be a selective antagonist i t to inhibit

by any natural PKIPBAN and most important was shown to from moths to a high 30-150 pmol). physiologically significant quantities on bioactivity of all of

above pcptidcs (amphiphilic pcptidcs, arc not i n this review arc by R.J.

i n i n this book.

llational

PIZIPBAN

PKPBAN

tar, receptor,

PKIPBAN littomlis melanization (Zheng al. 2007),

N-tc~minal

N-terminus/loops docking/interaction

therefore, ligantllreceptor (e.g.,

TM

rcceptor

proposed model Ihe the

ditTer

therelbre rcsults, li-om

(because ditTering

Ihe

the H. peltigera Ben-Aziz

furthcr receptor(s)

2.3.3

Design of Insect Control Agents

Receptor Cloning and Characterization

In order to gain additional SAR information studies focused also on the receptor. The studies in this part pursued two parallel avenues: (i) cloning of the receptor in order to be able to mutate it and to study its SAR and pharmacokinetic properties more deeply; and (ii) development of a binding assay, which could later be converted into an high throughput assay, for screening libraries of chemical, molecu-

and natural compounds in the search for small molecules that interact with the and which later might serve as insecticidelinsect-controlprototypes.

In pursuing this objective we recently cloned a receptor from S. larvae. which mediates cuticular evaluated the structure of its gene. The receptor exhibited high homology with the pheromone gland receptors, with differences in the region, the second outer loop and the third inner loop. Currently, the structural mechanisms of the interactions between ligands and GPCRs are still not clear, but the data on GPCRs that have been accumulated so far suggest that the extracellular are responsible for the ligand (Leff 1995). It is thus possible that the major difference observed in the N-terminal region of the PBAN-R may,

et and

account for optimal conspecific docking and interaction, which results in differing binding patterns of different ligands, elicitors), to the pheromone gland and larval receptors. The helices that are highly con-served between different PBAN-Rs might form a precise ligand interaction pocket which is required for the FXPRLa motif-induced conformational change and receptor activation.

The may explain the differences, (both within and between assays), between inhibitory patterns of BBC and those of the other antagonistic peptides, and may provide an insight into the nature (competitive or non-competitive) of the inhibitors. It is most likely that the common sequence of the PWPBAN elicitors (FXPRLa) docks in the highly conserved region of the receptors, whereas the other sequences of the elicitor molecule dock at different sites, which may among the various assays. In light of the above suggestions, we may further hypothesize that the various BBC and other antagonists bind outside the FXPRLa docking pocket, and may, be considered as non-competitive inhibitors, whose inhibitory activity

most likely, interference with the docking of the other parts of the elicitor ligand with its receptor. Since different ligand molecules may dock in different regions

of structural differences between the receptors), the degree of inhibition of a given elicitor imposed by a given BBC compound results in inhibitory potencies. We have previously suggested that BBC inhibitors are non-competitive, in light of the results of an in vitro radioisotope-receptor binding study carried out with

native receptor (Altstein and 2001) (see below) and based on the in vivo assays described above. A definite answer to all of the above questions will depend on evaluation based on SAR analysis by means of 3-D modeling of cloned and evaluation of their interactions with the various ligands and inhibitors. These issues are currently under investigation in our laboratory.

p:~rallel the hintling developctl gluntl H , pPltigcr.tr. The

(RRA) 'H-tyrosyl-PBAN28-33NH. as (Altstein Altstcin et nl. I999h): (he was

biotinylntctl (RpaPBANI-33NH.j usetl tagged ligand Yosef nI. 2009). microplotc

tlevelopment hindingissay. thc glantl

H. pplt ig~ln tlevelopetl, and the ntenihrane inct~hi~tion contlitions. (e.p., v;~lues, divalcnt ant1 protcase for

hinding tleterniinetl. The experitnental hc applictl of H'TS hintling

the conlbrmationally anrl nntagonists, hioassays that hccn ilevcl-

each of wily hctter entlogcnous n~echanisnis pcptitle fn~nil

prcscnt stutly. compounds [lie mcchi~nis~n.

regartling bioavnilahility (cuticulnr and well the the PKI

receptors mediate ma.jor (i.c.. selectivc. sti~hle ant1 cfkctive) itgonistic nntl

itntagonistic may. once hccome i~v:~ilable, hc cantlidates agrocheniiciil ant1 lield

ns the tlevelopment novel group cffec- live. cnvironnicntnlly Intlccd. o f the

have ctlreatly hasis tlesipn ant1 ol' ant1 compountls inhihitory ant1 hioavail- 'I'hese itre heing screenetl their rthility stirnulate

melanizrrtion S. littor-tllis larvae ancl hiosyn- H, y~1tigrrc.r. stutlies iuntl arc

by Nachman

Future

thc FXPRLa peptides yicltlcd interesting informittion the nature the PKIPBAN f a ~ n ~ l y pcpt~dcs,

target organ, 01' tramport, etc. however. about these pcptitlcs i~nresolvetl. especially rcgi~rtling nncl rcniains learned about chemical

hasis o f thc~r tlownstrcam ccllular species spcci ticity. rcccp- the mcchanisrns that untlerlay firnctional clivcrs~ty.

In to cloning of receptor. two assays were with the native pheromone PKIPBAN receptor of first was a radio-receptor assay in which was used a radio-lipand and Ben-Aziz 2001; second a microplate binding assay in which a PBAN was as a (Ben ct The assay was designed to facilitate its further into a HTS A method for obtaining an active receptor preparation from pheromone of the moth

was optimal preparation and buffer pH ions. inhihitors)

receptor-lipand were set up will to the cloned receptor for the development assays.

I n sutnniary. the availability of constrained selective and non-selective agonists and also of have oped for the above-mentioned functions has opened the to a understanding of the of the PKIPBAN y in moths and other insects. The knowledge gained in the course o f the regarding the effects of the on cndogcnous (native)

their penetration) their selectivity toward the various activities, as as the data on differing activity patterns of PBAN elicitors and on the structural changes in the that thcir activities. are all of importance for the design of additional. improved more potent. highly metabolically cost

compounds. which they for applications. After formulation preliminary cxperimcnts.

they could serve prototypes in of a of highly insect-specific and friendly insecticides. some

ahove antagonists been used as a for the synthesis novel improved with enhanced potency ability. compountls currently for to or inhibit cuticular in sex pheromone thesis in Results of these arc not detailed in this review summarized R.J. in another chapter in this book.

3 Concluding Remarks and Prospects

Intensive studies of on chemical and molecular origin. localization. many questions mode of action, much cellular activity. tor heterogeneity and

have of

route arc still

to bc

very of

In spite of this,

the structural, events.

thcir

thcir

thcir and

73

information e.g.,

most peptides

peptides ,..

conformationally high-aftinity

PWPBAN peptides

offer

a11 peptides

M.Sc. Aliza

0. (FXPRLamide) Helicoverlxt

Zen. Peptides al Dro.~oplriln mclnrro-

gflstcr. Soto peptide n improve

bioavailability. 60:

PKIPBAN J

NBssel Nerrmcierlce Systerns os Torgetsfor Pornsite ortd Pest Conrml.

Y, Neuroendocrine biosynthe- Heliotl~is pelrigera.

0, Heliotlris-l~eltigerfl:

Rational Design of Insect Control Agents

The that has already been accumulated and the tools, bioassays. in vitro binding assays, receptor-selective agonists and antagonists, receptor genes, etc.. that are currently available to us and to other laboratories offer great potential for further exploration of the above issues. Receptors are significant in understand-ing the biological function of any Np, especially in families where several exhibit similar bio-activities, and they play a central role in providing information on the direct correlation between the activity of a given Np and its target. Antagonists, especially those that are receptor-selective, such as the BBC described above, form excellent research tools for studying multi-peptide families that exhibit functional diversity. We anticipate that the availability of con-strained antagonists, the ligands that were developed in our laboratory. the cloned receptor and the binding assays that were developed, together with the in vivo bioassays will provide a solid basis for further studies that aim to gain a better insight into the mode of action of PBAN and the other in moths and other insects. Beyond the high scientific value of the above findings, the strategies and approaches that were developed in the course of the PBAN research also high potential for practical application, by providing a basis for generation of insect Np antagonist-based insect control agents, as described in this chapter.

Acknowledgements This research was supported by US-Israel Binational Agricultural Kesearch and Development Fund (BARD) (IS-3356-02). We would like to thank Professor Gilon of the Department of Organic Chemistry at the Hebrew University of Jerusalem, Israel for the design of the photo-affinity ligands and other linear and backbone cyclic Mrs. Orna Ben-Aziz and Mr. Michael Davidovitch for excellent experimental work and insect rearing. This manuscript forms part of the thesis of Hariton, a student at the Hebrew University of Jerusalem.

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