Clovamide, a Hydroxycinnamic Acid Amide, Is a Resistance Factor Against Phytophthora spp. in Theobroma cacao

Abstract

The hydroxycinnamic acid amides (HCAAs) are a diverse group of plant-specialized phenylpropanoid metabolites distributed widely in the plant kingdom and are known to be involved in tolerance to abiotic and biotic stress. The HCAA clovamide is reported in a small number of distantly related species. To explore the contribution of specialized metabolites to disease resistance in cacao (Theobroma cacao L., chocolate tree), we performed untargeted metabolomics using liquid chromatography – tandem mass spectrometry (LC-MS/MS) and compared the basal metabolite profiles in leaves of two cacao genotypes with contrasting levels of susceptibility to Phytophthora spp. Leaves of the tolerant genotype ‘Scavina 6’ (‘Sca6’) were found to accumulate dramatically higher levels of clovamide and several other HCAAs compared to the susceptible ‘Imperial College Selection 1’ (‘ICS1’). Clovamide was the most abundant metabolite in ‘Sca6’ leaf extracts based on MS signal, and was up to 58-fold higher in ‘Sca6’ than in ‘ICS1’. In vitro assays demonstrated that clovamide inhibits growth of three pathogens of cacao in the genus Phytophthora, is a substrate for cacao polyphenol oxidase, and is a contributor to enzymatic browning. Furthermore, clovamide inhibited proteinase and pectinase in vitro, activities associated with defense in plant-pathogen interactions. Fruit epidermal peels from both genotypes contained substantial amounts of clovamide, but two sulfated HCAAs were present at high abundance exclusively in ‘Sca6’ suggesting a potential functional role of these compounds. The potential to breed cacao with increased HCAAs for improved agricultural performance is discussed.

Mechanistic Insights into the Inhibition of SARS-CoV-2 Main Protease by Clovamide and Its Derivatives: In Silico Studies

“…The mechanisms (Scheme 3) were rendered likely by experimental evidence: upon radical scavenging (anti-or pro-oxidant effects), clovamide becomes oxidized to ortho-quinone species [43], which, then, readily react with thiol groups to form thiol adducts [42], forming the linkage between the sulfur atom of C145 and carbon on the quinone ring. More importantly, these quinone derivatives of clovamide can be naturally found in cacao as a result of both non-enzymatic and enzymatic oxidation mediated by PPO, which also involves a subsequent crosslinking with proteins [26,27]. In addition, clovamide has been determined to be a broad-spectrum enzyme inhibitor, shown to inhibit proteases by a generic mechanism involving oxidation to quinone and the formation of covalent linkages with reducing groups such as thiol [26].…”

“…The oxidized quinone species can then readily react with sulfur radicals or thiolate species of that thiol-containing cysteine residue [26]. Therefore, we investigated the three ortho-quinone derivatives (Figure 3) of clovamide to explore their potential reactivities with the C145 residue in forming covalent bonds.…”

“…We hypothesized that clovamide could act as a strong inhibitor to the main protease, for the following reasons: (1) Two catechol (o-hydroxy phenol) moieties present in the clovamide molecule, which are known to react with cysteine residues readily in oxidized states; when clovamide scavenges radical species, the catechol groups become oxidized into orthoquinones [24,25]. Alternatively, the oxidation and inhibition can be mediated by natural enzymatic browning or melanization processes, generating quinone species by polyphenol oxidase (PPO)-mediated oxidation and subsequent crosslinking with proteins [26,27]. The non-enzymatic oxidation of o-diphenol to quinone species of clovamide was also detected [26].…”

“…Alternatively, the oxidation and inhibition can be mediated by natural enzymatic browning or melanization processes, generating quinone species by polyphenol oxidase (PPO)-mediated oxidation and subsequent crosslinking with proteins [26,27]. The non-enzymatic oxidation of o-diphenol to quinone species of clovamide was also detected [26]. Quinones, particularly ortho-quinones, are shown to be reactive to thiolcontaining compounds which gives them potent antiviral activities [28,29].…”

“…The antiviral effect of clovamide was first reported by El-Sharawy et al (2017) against the H5N1 influenza A virus [31]. Recently, clovamide was suggested to be a broad-spectrum enzyme inhibitor adopting a generic mechanism involving the o-diphenol oxidation to its quinone form and indiscriminate covalent cross-linking to reducing groups, such as thiols or amines, in proteins [26].…”