(120) Assay Card

General Information
Summary A range of natural variants were selected based on net charge and/or hydrophobicity and were tested alongside the prototypic cyclotide kB1 in larval development assays with H. contortus and T. colubriformis.
Condition 200 uL of agar (2 %) was deposited into each well of a 96-well microtitre plate. After this had solidified, nematode egg solution (30 uL) was added into each well. Water (10 uL) was added to the control wells and cyclotide solution (10 uL, varying concentrations) was added into the treatment wells. The final concentration range was 0.3-33.3 ug mL-1 (0.09-11.53 uM). After 24 h, growth medium (20 uL) was added to each well. The growth medium consisted of Earles salt solution (10 %, v/v), yeast extract (1 %, w/v), sodium bicarbonate (1 mM) and saline solution (0.9 % sodium chloride, w/v). The nematodes were allowed to feed and develop for four days and then killed using Lugols iodine solution and scored for the number of fully developed infective stage larvae (L3) present in each well. Each treatment was conducted in at least triplicate and controls for water and/or ethanol (20 %) were included in each assay as required.
Result In summary, the most potent cyclotides tested were all extracted from V. odorata and contained three or four basic amino acids. The IC50 values for this group were significantly lower than all other cyclotides that contained fewer (0-2) basic amino acids. Acetylation of the lysine side chains dramatically decreased the anthelmintic activity as evidenced by the IC50 value of 2.30 M, compared to 0.13 M for native cO2 (18-fold decrease). The IC50 value for cO2-Glu(Me) was 0.76 M, representing an approximately sixfold decrease in potency. Similar decreases in activity were observed with T. colubriformis
AssayType Nematocidal

References
Colgrave ML, Kotze AC, Ireland DC, Wang CK, Craik DJ (2008) The anthelmintic activity of the cyclotides: natural variants with enhanced activity Chembiochem 9:1939-1945

Cross-references
Proteins Assayed kalata B1
kalata S
cycloviolacin O12
cycloviolacin O1
cycloviolacin O13
cycloviolacin O16
cycloviolacin O24
cycloviolacin O2
cycloviolacin O14
cycloviolacin O15
kalata B2
vhl-1
cycloviolacin Y1
cycloviolacin Y4
cycloviolacin Y5
kalata B7
kalata B6
cycloviolacin O3
cycloviolacin O8
cycloviolacin H3
[Lys(Ac)]2cyO2
[Glu(Me)]cyO2