title: Dihydroisoxazole inhibitors of Anopheles gambiae seminal transglutaminase AgTG3.
abstract: BACKGROUND
  Current vector-based malaria control strategies are threatened by the rise of biochemical and behavioural resistance in mosquitoes. Researching mosquito traits of immunity and fertility is required to find potential targets for new vector control strategies. The seminal transglutaminase AgTG3 coagulates male Anopheles gambiae seminal fluids, forming a 'mating plug' that is required for male reproductive success. Inhibitors of AgTG3 can be useful both as chemical probes of A. gambiae reproductive biology and may further the development of new chemosterilants for mosquito population control.
  
  
  METHODS
  A targeted library of 3-bromo-4,5-dihydroxoisoxazole inhibitors were synthesized and screened for inhibition of AgTG3 in a fluorescent, plate-based assay. Positive hits were tested for in vitro activity using cross-linking and mass spectrometry, and in vivo efficacy in laboratory mating assays.
  
  
  RESULTS
  A targeted chemical library was screened for inhibition of AgTG3 in a fluorescent plate-based assay using its native substrate, plugin. Several inhibitors were identified with IC50 < 10 μM. Preliminary structure-activity relationships within the library support the stereo-specificity and preference for aromatic substituents in the chemical scaffold. Both inhibition of plugin cross-linking and covalent modification of the active site cysteine of AgTG3 were verified. Administration of an AgTG3 inhibitor to A. gambiae males by intrathoracic injection led to a 15% reduction in mating plug transfer in laboratory mating assays.
  
  
  CONCLUSIONS
  A targeted screen has identified chemical inhibitors of A. gambiae transglutaminase 3 (AgTG3). The most potent inhibitors are known inhibitors of human transglutaminase 2, suggesting a common binding pose may exist within the active site of both enzymes. Future efforts to develop additional inhibitors will provide chemical tools to address important biological questions regarding the role of the A. gambiae mating plug. A second use for transglutaminase inhibitors exists for the study of haemolymph coagulation and immune responses to wound healing in insects.
authors.forename: Binh V
authors.initials: BV
authors.lastname: Le
authors.forename: Cornelius
authors.initials: C
authors.lastname: Klöck
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schatz
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Nguyen
authors.forename: Evdoxia G
authors.initials: EG
authors.lastname: Kakani
authors.forename: Flaminia
authors.initials: F
authors.lastname: Catteruccia
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
authors.affiliation: Department of Chemistry, Yale University, P,O, Box 208107, New Haven, CT 06520-8107, USA. richard.baxter@yale.edu.
authors.forename: Richard H G
authors.initials: RH
authors.lastname: Baxter
mesh_terms: D000818:Animals; D000852:Anopheles / Q000201:enzymology*; D020134:Catalytic Domain; D002629:Chemosterilants / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D019476:Insect Proteins / Q000037:antagonists & inhibitors*; D007555:Isoxazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D008297:Male; D008958:Models, Molecular; D015394:Molecular Structure; D009032:Mosquito Control / Q000379:methods*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000187:drug effects; D012661:Semen / Q000201:enzymology*; D054852:Small Molecule Libraries; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D011503:Transglutaminases / Q000037:antagonists & inhibitors*
chemical_list: D002629:Chemosterilants; D003432:Cross-Linking Reagents; D015853:Cysteine Proteinase Inhibitors; D019476:Insect Proteins; D007555:Isoxazoles; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; C498712:TGM3 protein, human; D011503:Transglutaminases