Variables básicas y parámetros del terreno

3.4.3.1 Metabolism of Insecticides

To assess the metabolic profiles of the different CYP6P9a and CYP6P9b proteinswith different classes of insecticides (Types I and II pyrethroids, etofenprox, bendiocarb, propoxur, DDT and malathion), metabolism assay was conducted using HPLC. Both CYP6P9a and CYP6P9b metabolizes Type I and Type II pyrethroids, with statistically significant depletion (+NADPH vs –NADPH) obtained from all proteins from resistant alleles compared with corresponding proteins from FANG. No significant activities were obtained against DDT, bendiocarb, propoxur and malathion), consistent with the molecular docking predictions as well as probes substrates analysis, in which no activities were respectively predicted and/or obtained against bendiocarb, propoxur and DDT.

Figure 3.14: Correlation between IC50 of test insecticide inhibitors on MOZCYP6P9b bmetabolism

of DEF and the ChemScore values from docking with GOLD. IC50 values vs Chemscore for

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CYP6P9a Alleles:

Recombinant proteins expressed from alleles of resistant strains produced higher activities against pyrethroid insecticides compared with FANGCYP6P9a. These differences were statistically significant (p<0.05) for all pyrethroid insecticides. Highest activities were obtained from MALCYP6P9a which deplete 65.38%±1.5, 60.48%±2.65, 68.38%±1.83, 75.41%±1.72 and 46.04%±1.26 each of permethrin, bifenthrin, deltamethrin, λ-cyhalothrin and etofenprox, respectively (Table 3.16 and Figure 3.15A). In contrast, FANGCYP6P9a with lowest activities deplete only 29.0%±1.42, 16.6%±5.41, 18.11%±2.5, 15.46%±4.43 and 7.28%±1.53 each of permethrin, bifenthrin, deltamethrin, λ-cyhalothrin and etofenprox, respectively. This clearly indicated that the protein from the susceptible allele, FANG is a poor metaboliser of pyrethroid insecticides compared with those from the resistant alleles. Table 3.16: Percentage depletion of pyrethroid insecticides by recombinant CYP6P9a and CYP6P9b Recombinant Proteins Permethrin Bifenthrin Deltamethrin λ-cyhalothrin Etofenprox

FANGCYP6P9a 29.0±1.42 16.6±5.41 18.11±2.15 15.46±4.43 7.28±1.53 UGANCYP6P9a 60.71±0.92* 50.31±3.8* 57.9±2.74* 47.74±1.5* 42.55±1.03* BENCYP6P9a 66.42±2.23* 51.02±0.75* 59.54±5.11* 52.45±1.74* 41.22±1.54* MALCYP6P9a 65.38±1.5* 60.48±2.65** 68.38±1.83** 75.41±1.72* 46.04±1.26* FANGCYP6P9b 13.7±4.23 22.38±5.08 6.2±1.5 15.49±3.13 16.88±3.06 UGANCYP6P9b 88.58±3.48** 88.8±1.61** 62.53±4.04** 78.76±1.31** 57.91±1.55* BENCYP6P9b 89.63±0.63** 89.19±1.01** 62.03±1.14** 49.35±4.41* 37.15±4.15* MOZCYP6P9b 91.6±2.5** 81.6±0.25** 81.69±2.27** 86.51±1.14** 71.59±1.42**

Values are mean ± S.D. of three replicates compared with negative control (-NADPH);

* and **Significantly different from FANGCYP6P9a or FANGCYP6Pb at p<0.05 or p<0.01 respectively.

CYP6P9b Alleles:

Recombinant proteins from CYP6P9b of resistant strains metabolizes pyrethroid insecticides with higher activities several fold more than the protein from susceptibe allele, FANGCYP6P9b (Table 3.16 and Figure 3.15B). These differences were statistically significant with the southern African allele MOZCYP6P9b having highest activity: 91.6%±2.5, 81.6%±0.25, 81.69%±2.27, 86.51%±1.14, 71.59%±1.42 depletion, respectively for permethrin, bifenthrin, deltamethrin, λ-cyahalothrin and etofenprox, compared with FANGCYP6P9b which depleted only 13.7±4.23, 22.38%±5.08, 6.2%±1.5,

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15.49%±3.13 and 16.88%±3.03 of these insecticides. The differences between the resistant and susceptible alleles possibly explained the reason why FANG strain is susceptible to pyrethroids.

As observed with CYP6P9a, recombinant protein from the southern African allele of CYP6P9b (MOZCYP6P9b) has the highest activity consistent with the highest pyrethroid resistance reported in this region. However, as in fluorescent probes assay, CYP6P9b proteins exhibited higher activities against pyrethroids compared with CYP6P9a, confirming that CYP6P9b is more efficient metaboliser than CYP6P9a. MOZCYP6P9b has the highest activity of all the alleles screened with pyrethroids is not surprising for MOZCYP6P9b protein was predicted to have the highest activity of all alleles, and has been established as having high activity with probe substrates and portrayed the tightest binding to Type I and Type II pyrethroids from inhibition assay.

3.4.3.2 Kinetics Analysis with Permethrin and Deltamethrin

Kinetics analysis was carried out with recombinant protein variants of four alleles each of CYP6P9a and MOZCYP6P9b, in order to establish the catalytic constant (Kcat) and Michaelis constant

Figure 3.15: Percentage depletion of 20µM pyrethroids by recombinant CYP6P9a(A) and CYP6P9b (B).Results are average of three replicates (n = 3) compared with negative control (-NADPH).

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(KM) for permethrin and deltamethrin. The Kcat provides information on the speed with which different

protein variants metabolise (clear) insecticide toxicant in vitro, while the KM will highlight the affinity of

each allele toward the insecticides tested. These parameters can help in assessing any differences in metabolic efficiencies between the alleles from resistant strains and those from FANG. Steady-state kinetic parameters for CYP6P9a and CYP6P9b with permethrin and deltamethrin are given in Table 3.17. Reactions followed Michaelis-Menten pattern (Figure 3.16) with KM values within ranges (1-

50µM) described for binding and metabolism of pyrethroids by insect cDNA-expressed P450s (Stevenson et al., 2012, Stevenson et al., 2011) and lower than KM values obtained from An. minimus’

CYP6P7 and CYP6AA3 with pyrethroids (Duangkaew et al., 2011). Kcat values obtained were also within

the broad ranges recorded in the literature (1-20min-1) for the activities of insect P450s with pyrethroids (Stevenson et al., 2012) but higher than established for some cDNA-expressed P450s, including An. gambiae CYP6M2, Ae. aegypti CYP9J families, as well as An. gambiae CYP6P3 with permethrin (Stevenson et al., 2011, Stevenson et al., 2012, Muller et al., 2008).

Figure 3.16: Michaelis-Menten plot of permethrin (A) and (B) and deltamethrin (C) and (D) metabolism by recombinant CYP6P9a and CYP6P9a. Each point is a mean±S.E.M. of three replicates.

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CYP6P9a Alleles:

Kinetic parameters obtaind revealed that CYP6P9a proteins from the resistant alleles possess higher Kcat and higher affinity (low KM) compared with proteins expressed from the susceptible FANG.

The Kcat of UGANCYP6P9a and MALCYP6P9a with permethrin are two- and three-fold higher than

values obtained from FANGCYP6P9a, respectively (Table 3.17), and that of BENCYP6P9a and MALCYP6P9a with deltamethrin are as well two- and three-fold higher compared with the Kcat

obtained from FANGCYP6P9a. These differences were statistically significant (p<0.05) for BENCYP6P9a and MALCYP6P9a compared with FANGCYP6P9a. In terms of KM, for permethrin, statistically significant

differences were also obtained with FANGCYP6P9aon average having half the affinity (KM values two-

fold higher) compared with the CYP6P9a from resistant alleles. This translated into statistically significant differences (ANOVA Tukey’s test of significant at p<0.05) in terms of efficiency with BENCYP6P9a and UGANCYP6P9a having catalytic efficiencies for permethrin (three-fold higher) than FANGCYP6P9a while MALCYP6P9a exhibited catalytic efficiency for permethrin six-fold higher than FANGCYP6P9a. While FANGCYP6P9a exhibited higher affinity for deltamethrin than permethrin, the CYP6P9a from resistant alleles showed comparable KM for both permethrin and deltamethrin. As a

result of these differences the catalytic efficiencies of BENCYP6P9a and UGANCYP6P9a for deltamethrin were on average three-fold the values from FANGCYP6P9a while the Kcat for

deltamethrin from MALCYP6P9a was four-fold higher than values from FANGCYP6P9a (Figure 3.17).

CYP6P9b Alleles:

For permethrin CYP6P9b alleles from resistant individuals produced higher Kcat values

compared with FANGCYP6P9b; these statistically significant differences ranged from twice the Kcat of FANGCYP6P9b as obtained from UGANCYP6P9b, to three-fold higher Kcat as observed from MOZCYP6P9b, to four-fold higher Kcat as obtained from BENCYP6P9b (Table 3.17).

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Table 3.17: Kinetic Constants for Recombinant CYP6P9a- and CYP6P9b Permethrin and Deltamethrin Metabolism

Recombinant Proteins Kcat (min-1) KM (µM) Kcat/KM (min-1 µM-1)

Permethrin BENCYP6P9a 9.37±3.32a 19.02±3.13c 0.49±0.19* UGANCYP6P9a 7.35±1.02 20.11±3.91c 0.37±0.08* FANGCYP6P9a 4.95±1.92 36.25±15.22 0.14±0.07 MALCYP6P9a 15.41±6.30a 18.77±11.76c 0.82±0.06* BENCYP6P9b 15.91±5.45b 21.94±7.97 0.73±0.36$ UGANCYP6P9b 8.61±3.40b 21.22±10.32 0.41±0.3$ FANGCYP6P9b 4.5±1.35 20.47±8.31 0.21±0.11$ MOZCYP6P9b 12.38±4.5b 12.68±2.68d 0.97±0.41$ Deltamethrin BENCYP6P9a 8.78±2.62a 15.67±4.64c 0.56±0.23* UGANCYP6P9a 7.96±3.62 14.48±3.58c 0.54±0.28* FANGCYP6P9a 4.87±2.06a 24.03±3.12 0.20±0.08 MALCYP6P9a 14.65±4.12a 18.26±6.97 0.80±0.38* BENCYP6P9b 13.63±5.40b 15.98±5.63 0.85±0.45$ UGANCYP6P9b 10.44±4.00b 12.97±4.96d 0.80±0.35$ FANGCYP6P9b 4.92±0.82 19.5±5.53 0.25±0.08 MOZCYP6P9b 12.09±1.44b 9.9±1.65d 1.22±0.25$ BENCYP6P9a 8.78±2.62a 15.67±4.64c 0.56±0.23*

Values are mean ±S.D. of three replicates

Apparent Kcat was calculated as pmol/min/pmol P450; Catalytic efficiency was calculated as Kcat/KM a,b

Significantly different Kcat values compared withFANGCYP6P9a and FANGCYP6P9b respectively, p<0.05. c,d

Significantly different KM values compared with FANGCYP6P9a and FANGCYP6P9b respectively, p<0.05.

*,$ Significant differences between Kcat/KM values, respectively compared withFANCYP6P9a and FANGCYP6P9b, p<0.05.

Same pattern was obtained with deltamethrin, with UGANCYP6P9b having two-fold Kcat

compared with FANGCYP6P9b, while BENCYP6P9band MOZCYP6P9b exhibited Kcat values on average

three-fold that of FANGCYP6P9b. No major difference in terms of KM for permethrin and deltamethrin

were observed between recombinant proteins from susceptible and resistant alleles of CYP6P9b, with the exception of UGANCYP6P9b with deltamethrin, and the southern African MOZCYP6P9b which consistently exhibited lowest, reproducible KM both for permethrin and deltamethrin). These low KM

values from MOZCYP6P9b protein showed that the MOZCYP6P9b allele occupies a central position in terms of pyrethroid metabolism and the high affinity it exhibited, which is consistent with the molecular docking simulations in which the MOZCYP6P9b portrayed multiple, productive binding conformations, as well as fluorescent probes assay from which recombinant MOZCYP6P9b portrayed a

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very high activity with the probe substrate diethoxyfluorescein, as well as tightest binding with pyrethroids, especially the Type II class, as obtained from inhibition assay.

For permethrin, UGANCYP6P9b, BENCYP6P9b and MOZCYP6P9b were calculated as having two-fold, three-fold and five-hold higher catalytic efficiencies respectively compared with FANGCYP6P9b (Figure 3.17). With deltamethrin, BENCYP6P9b and UGANCYP6P9b possess catalytic efficiencies more than three-fold higher than FANGCYP6P9b, while MOZCYP6P9b with the highest catalytic efficiency of 1.22 min-1µM-1±0.25 is five times more efficient in deltamethrin metabolism than the recombinant FANGCYP6P9b from the susceptible allele.

In agreement with the results from fluorescent probes assays and initial testing of depletion of pyrethroids, recombinant CYP6P9b exhibited higher activities compared with their corresponding CYP6P9a, reflecting the central position of CYP6P9b in pyrethroid metabolism and resistance, especially MOZCYP6P9b with highest intrinsic clearance (CLint) for both permethrin and deltamethrin.

Figure 3.17: 4D plot of the kinetic constants and catalytic efficiencies of recombinant proteins of CYP6P9a and CYP6P9b with (A) permethrin and (B) deltamethrin. x-axis is the Kcat (min-1); y-axis is the KM (µM); catalytic efficiency is calculated as x/y (min-1 µM-1).

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