Features of interactions responsible for antifungal activity against resistant type cytochrome bc1: A data-driven analysis based on the binding free energy at the atomic level

Abstract

Quinone outside inhibitors (QoIs), which inhibit the mitochondrial respiratory system by binding to the Qo site of Complex III in fungi, are widely used as pesticides with broad spectrum antifungal activity. However, excessive use of QoIs leads to pesticide resistance through mutation of amino acid residues in the Qo site. Recently, metyltetraprole, a novel QoI that is effective against wild-type and resistant mutant fungi, was developed. Interestingly, metyltetraprole has a very similar structure to other QoIs, azoxystrobin and pyraclostrobin, which do not act on resistant mutants. However, it is unknown how slight structural differences in these inhibitors alter their effectiveness towards fungi with amino acid mutations in the Qo site of Complex III. Therefore, we studied the features of interactions of inhibitors effective towards resistant mutants by quantitatively comparing the interaction profiles of three QoIs at the atomic level. First, we reproduced the binding affinity by the thermodynamic integration (TI) method, which treated explicitly environmental molecules and considered the pseudo-binding pathway. As such, a good correlation (R2 = 0.74) was observed between the binding free energy calculated using the TI method and experimentally observed pIC50 value in 12 inhibitor-target pairs, including wild-type and mutant Complex III in two fungal species, Zymoseptoria tritici and Pyrenophora teres. Trajectory analysis of this TI calculation revealed that the effectiveness against resistant mutant fungi strongly depended on the interaction of constituent parts of the inhibitor disposed near the active center of the target protein. Specifically, the key in the effectiveness against resistant mutant fungi is that the corresponding component part, tetrazolinone moiety of metyltetraprole, traded off Coulomb and van der Waals interactions in response to subtle changes in the binding pose.

The cytochrome bc1 complex as an antipathogenic target

“…The compound has side chain similar to that of the synthetic strobilurin pyraclostrobin, but has a unique tetrazolinone moiety. It is suggested that this tetrazolinone moiety will not form the same highly specific interactions with the Q o site as do other strobilurin‐based QoIs and in consequence can accommodate changes in the target, such as G143A [64]. Thus, the steric clash due to G143A that can compromise the binding of QoIs would be limited by the unique size and shape of the tetrazolinone.…”

Antifungal activity of metyltetraprole against the existing QoI‐resistant isolates of various plant pathogenic fungi

“…Although metyltetraprole itself is a highly promising agricultural fungicide, which is expected to be commercialized in the near future (https://sumitomo-chem-agro.com/blog/sumitomo-chemical-submits-a-registration-application-in-the-eu-for-metyltetraprole-a-new-fungicide/), its discovery has paved the way to overcome the mutations G143A and F129L in the history of synthetic QoIs. However, the numbers of tested isolates and pathogen species were limited in our previous reports …”

“…This second type of QoI-R mutants have been reported to show partial resistance to QoI in pathogenic fungi such as Pyrenophora teres etc. 1,2 Metyltetraprole, 1-(2-{[1-(4-chlorophenyl)-1H-pyrazol-3-yl]oxy methyl}-3-methylphenyl)-1,4-dihydro-4-methyl-5H-tetrazol-5-one, is a novel fungicide with the unique tetrazolinone pharmacophore found by Sumitomo Chemical Co. Ltd. [5][6][7][8] Its molecule has a side chain very similar to that of pyraclostrobin, a representative of the existing QoIs, and it also binds to the Qo site of the cytochrome bc1 complex. 5,9 A competitive binding assay using radiolabeled metyltetraprole and pyraclostrobin showed that both fungicides bound to the same target pocket in the cytochrome bc1 complex.…”

“…However, the numbers of tested isolates and pathogen species were limited in our previous reports. 7,8 In the case of QoIs, it has been reported that the orthologous amino acid mutations result in similar resistance profiles across different organisms with respect to the strength of resistance; the G143A mutation causes very high level of resistance (the resistance factor RF, [EC 50 of the resistant mutant]/[EC 50 of the wild type], is always greater than 100) to all commercial QoIs in any known cases of more than 20 pathogen species. 1,2 However, recently some cases of different resistance profiles within the orthologous mutations of different pathogen species were reported in cases of resistance against succinate dehydrogenase inhibitor (SDHI), another important agricultural fungicide class.…”

Caracterização da sensibilidade de >i<Alternaria alternata>/i<, agente causal da mancha marrom em tangerinas, a fungicidas inibidores da quinona externa

“…Trabalhos realizados por Mondal (2005), Pasche et al (2007) e Bradley e Pedersen (2011) relatam diferenças de até dez vezes nas doses necessárias para a inibição de 50% da germinação de conídios entre piraclostrobina e azoxistrobina. Arakawa et al (2018) avaliaram as características responsáveis pela interação entre três diferentes inibidores da quinona externa em indivíduos selvagens e resistentes, a fim de compreender os fatores responsáveis pela sua efetividade ou perda da mesma em casos onde há mutações genéticas responsáveis por conferir resistência. Apesar de serem do mesmo grupo químico, as moléculas apresentam componentes estruturais diferentes e revelaram que a eficácia contra fungos mutantes resistentes dependia fortemente da interação de partes constituintes do inibidor disposto próximo ao centro ativo da proteína alvo, neste caso o citocromo b.…”