LDAs were conducted in a microtiter plate format. Briefly, 200 µL of 2% agar was deposited into each well of a 96-well microtiter plate. After this had solidified, 30 µL of nematode egg solution was aliquotted into each well. Water (10 µL) was added to the control wells, while 10 µL of cyclotide solution (varying concentrations) was aliquotted into the treatment wells. The final concentration range was 1.0?62.5 µg/mL (0.3?21.5 µM) for LDAs. For each assay, triplicate wells were examined at each concentration. The plates were incubated overnight at 28 °C and the unhatched eggs and L1 larvae present in each well were counted.
For the LDA, plates were set up as described above, incubated overnight at 28 °C, and then 20 µL of growth medium was added to each well. The growth medium consisted of Earle’s salt solution (10% v/v), yeast extract (1% w/v), sodium bicarbonate (1 mM), and saline solution (0.9% sodium chloride, w/v) (24). The nematodes were allowed to feed and develop for four days and then killed using Lugol’s 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 or 20% ethanol were included in each assay as required.
A range of cyclotides was tested, including the prototypic cyclotide kalata B1, as well as kalata B2, B3, B5, B6, and B7 isolated from Oldenlandia affinis. The natural variants were found to have differing larvicidal activities, with kalata B6 the most potent of the variants tested having an IC50 of 2.6 µg/mL for H. contortus and 7.9 µg/mL for T. colubriformis kB1 and kB2 showed comparable activities (around 5?6 µg/mL for H. contortus and 17?21 µg/mL for T. colubriformis), while kB7 had markedly reduced activity (ca. 17?19 µg/mL for H. contortus and T. colubriformis) and kB3 showed very little activity (IC50 could not be calculated). kB5 was only tested at three concentrations (owing to limited material) within the dose response curve for kB1 and showed comparable activity at these concentrations. The percentage inhibition of development resulting from treatment with kB3 was <5% (H. contortus) at 12.5 µg/mL and only 18% (T. colubriformis) at 20 µg/mL, concentrations at which ~90% inhibition was observed for kB1 and 100% inhibition for kB6. Typically, T. colubriformis were 2-fold less sensitive to a given cyclotide than H. contortus.
The Ala-scan study showed that the cyclotide mutants could be subdivided into groups according to their potency as inhibitors of larval development. The data used to delineate groups was derived from assays with the higher level of cyclotide with each nematode species, but the general pattern was the same at the lower concentration. The groupings were (1) inhibition similar to the wild-type (kB1): G7A, T9A in loop 2; G14A in loop 3; T16A in loop 4; S18A in loop 5; and T23A, N25A, G26A in loop 6; (2) inhibition at 65?90%: P13A in loop 3; and L27A, V29A in loop 6; (3) inhibition at 25?40%: N11A, T12A in loop 3; V21A in loop 5; and R24A and P28A in loop 6; and (4) inhibition < 25%: G2A, E3A, T4A in loop 1; and V6A, G8A in loop 2.
An all-D-isomer of kalata B1 was also tested and shown to have comparable activity to its native parent