US 20060178292A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0178292 A1 Ruvkun et al. (43) Pub. Date: Aug. 10, 2006 (54) METHODS AND COMPOSITIONS 0F Related Us, Application Data ECDYSOZOAN MOLT INHIBITION (60) Provisional application No. 60/437,235, ?led on Dec. (75) Inventors: Gary Ruvkun, Newton, MA (US); 31, 2002. Alison Frand, Cambridge, MA (US) Publication Classi?cation Correspondence Address: CLARK & ELBING LLP (51) IIlt- Cl 101 FEDERAL STREET A01N 37/18 (2006.01) BOSTON, MA 02110 (US) C12Q 1/68 (2006.01) G01N 33/567 (2006.01) G01N 33/53 (2006.01) (73) Assignee: The General Hospital Corporation, (52) US. Cl. ................................... .. 514/2; 435/6; 435/7.2 Boston, MA (US) (57) ABSTRACT (21) Appl_ No; 10/540,445 In general, this invention relates to nucleic acid and amino acid sequences involved in molting and the use of these (22) PCT Filed; Dec 31, 2003 sequences as targets for the development of compounds that disrupt EcdysoZoan molting, and are useful as insecticides, (86) PCT No.1 PCT/US03/41788 nematicides, and anti-parasitic agents. Patent Application Publication Aug. 10, 2006 Sheet 1 0f 5 US 2006/0178292 A1 Patent Application Publication Aug. 10, 2006 Sheet 2 0f 5 US 2006/0178292 A1 FIGURE 2 % ?uorescent O % Fluorescent ' SP.»:5: 3:;mIll 0.En Patent Application Publication Aug. 10, 2006 Sheet 3 0f 5 US 2006/0178292 A1 FIGURE 3 .ammpumwi mm?5 . omu Patent Application Publication Aug. 10, 2006 Sheet 4 0f 5 US 2006/0178292 A1 FIGUREQ E35050». a64 __ 'hhra23- awn-.37 .m1t— 1'2 qhg-rf mIt-16 unit-13 Vector NHR AGE hint FB'IN 43 45' ~30‘ 282' 2'1 57 4O 42' 32 39 26 1 EmowQoEtB 08642 RNAi Patent Application Publication Aug. 10, 2006 Sheet 5 0f 5 US 2006/0178292 A1 FIGURE 5 9.6%:115=Lm....m€ US 2006/0178292 A1 Aug. 10, 2006 METHODS AND COMPOSITIONS OF compound, Where an alteration in expression identi?es the ECDYSOZOAN MOLT INHIBITION candidate compound as a candidate compound that modu lates molting. STATEMENT AS TO FEDERALLY SPONSORED [0008] In various embodiments of the previous aspects, RESEARCH the method identi?es a compound that increases or decreases [0001] This Work Was supported in part by the National transcription of a mlt nucleic acid molecule. In other Institutes of Health (NIH GM 44619). The government may embodiments of the previous aspects, the method identi?es have certain rights in this invention. a compound that increases or decreases translation of an mRNA transcribed from the mlt nucleic acid molecule. In BACKGROUND OF THE INVENTION still other embodiments of the identi?cation methods described herein, the compound is a member of a chemical [0002] In general, the invention features methods and library. In preferred embodiments, the cell is in a nematode. compositions that disrupt molting and are therefore useful targets for pesticides. [0009] Typically, a compound that decreases transcription or translation of a mlt nucleic acid molecule is useful in the [0003] Nematodes represent one out of every ?ve animals invention. For some applications, hoWever, a compound that on the planet, and virtually all plant and animal species are increases transcription or translation of a mlt nucleic acid targeted by at least one parasitic nematode. Plant-parasitic molecule is useful, for example, a mlt nucleic acid (e.g., nematodes reduce the yield of the World’s 40 major food W08F4.6, F09B12.1, or W01F3.3) that When overexpressed staples resulting in losses of approximately 12.3% annually. leads to larval arrest or death, or a mlt nucleic acid (e.g., Parasitic nematodes also damage human and domestic ani C17G1.6, CD4.6, C42D8.5, F08C6.1) that encodes a mal health. Lymphatic ?lariasis and elephantiasis are among secreted protease, Which degrades EcdysoZoan cuticle and the most devastating human tropical diseases. The World leads to larval arrest or death. Health Organization estimated that these diseases affected 120 million people WorldWide in 1992. [0010] In a related aspect, the invention provides yet another method for identifying a candidate compound that [0004] The impact of nematodes on human, animal, and disrupts molting in an EcdysoZoan. The method involves (a) plant health has resulted in the search for effective nemati providing a cell expressing a MLT polypeptide; (b) contact cides. BenZimidaZoles and avermectins are tWo common ing the cell With a candidate compound; and (c) comparing nematicides, Which target microtubule assembly and muscle the biological activity of the MLT polypeptide in the cell activity, respectively. Unfortunately, resistance to these contacted With the candidate compound to a control cell not compounds is increasingly common. In addition, these com contacted With said candidate compound, Where an alter pounds can have toxic effects on humans and other animals. ation in the biological activity of the MLT polypeptide Moreover, these nematicides are not effective against all identi?es the candidate compound as a candidate compound nematodes. Thus more effective and speci?c nematicides are that disrupts molting. required. [0011] In various embodiments, the cell is a nematode cell SUMMARY OF THE INVENTION or a mammalian cell. In other embodiments, the MLT polypeptide is a protease. In still other embodiments, the [0005] The present invention features improved methods biological activity of MLT polypeptide is monitored With an and compositions for inhibiting molting in EcdysoZoans, enZymatic assay or an immunological assay. In other pre including nematodes, parasitic nematodes, and insects. ferred embodiments, the cell is in a nematode and the biological activity is monitored by detecting molting. [0006] In one aspect, the invention provides a method for identifying a candidate compound that disrupts molting in an [0012] In another related aspect, the invention provides EcdysoZoan (e.g., an insect or nematode). The method yet another method for identifying a candidate compound includes the steps of: (a) providing a cell expressing a mlt that disrupts molting. The method includes the steps of: (a) nucleic acid molecule or an ortholog of a mlt nucleic acid contacting a nematode With a candidate compound; and (b) molecule; (b) contacting the cell With a candidate com comparing molting in the nematode contacted With the pound; and (c) comparing the expression of the mlt nucleic candidate compound to a control nematode not contacted acid molecule in the cell contacted With the candidate With said candidate compound, Where an alteration in molt compound With the expression of the nucleic acid molecule ing identi?es the candidate compound as a candidate com in a control cell not contacted With said candidate com pound that disrupts molting. pound, Where an alteration in expression identi?es the candidate compound as a candidate compound that disrupts [0013] In yet another related aspect, the invention pro molting. vides a yet further method of identifying a candidate com pound that disrupts EcdysoZoan molting. The method [0007] In a related aspect, the invention provides another includes the steps of: (a) contacting a cell containing a mlt method for identifying a candidate compound that disrupts nucleic acid regulatory region fused to a detectable reporter molting in a nematode. The method includes the steps of: (a) gene With a candidate compound; (b) detecting the expres providing a nematode cell expressing a mlt nucleic acid sion of the reporter gene; and (c) comparing the reporter molecule; (b) contacting the nematode cell With a candidate gene expression in the cell contacted With the candidate compound; and (c) comparing the expression of the mlt compound With a control cell not contacted With the candi nucleic acid molecule in the cell contacted With the candi date compound, Where an alteration in the expression of the date compound With the expression of the nucleic acid reporter gene identi?es the candidate compound as a can molecule in a control cell not contacted With said candidate didate compound that disrupts molting. US 2006/0178292 A1 Aug. 10, 2006 [0014] In various embodiments of the previous aspect, the that inhibits molting (e.g., C01H6.5, C17G1.6, C45B2.7, alteration is an alteration in the timing of reporter gene F11C1.6, F18C12.2, F29D11.1, F53G12.3, F56C11.1, expression of at least 10%, 20%, 30%, 40%, 50%, 60%, or K04F10.4, T05C12.10, T27F2.1, Y23H5A.7, and ZK270.1). even 70%, 80%, 90%, 95%, or 99% relative to the timing of In other embodiments, the naturally occurring mlt nucleic expression in a control nematode not contacted With the acid encodes a component of a secretory pathWay (e.g., candidate compound. In another embodiment, the alteration ZK1014.1, H15N14.1, F26H9.6, Y63D3A.5, C56C10.3, is an alteration in the level of expression of the reporter gene ZK180.4, F57H12.1, C39F7.4, Y113G7A.3, R160.1, of at least 10%, 20%, 30%, 40%, 50%, 60%, or even 70%, C02C6.1, E03H4.8, F59E10.3, K12H4.4, D1014.3, 80%, 90%, 95%, or 99% relative to the level of expression C13B9.3, F43D9.3). In other embodiments, the naturally in a control nematode not contacted With the candidate occurring mlt nucleic acid encodes a protein that functions compound. In another embodiment, the alteration is an in protein synthesis (e.g., B0336.10, B0393.1, C04F12.4, alteration in the cellular expression pattern of the reporter C23G10.3, D1007.6, F28D1.7, F35H10.4, F37C12.11, gene relative to the cellular expression pattern in a control F37C12.9, F40F11.1, F53A3.3, T01C3.6, T05F1.3, nematode not contacted With the candidate compound. Y45F10D.12). In still other embodiments, the inactivation [0015] In another related aspect, the invention provides a or inhibition of a naturally occurring mlt nucleic acid method for identifying a candidate compound that disrupts produces mlt defects in less than 5% of larvae (e.g., EcdysoZoan molting.