Staphyloxanthin Inhibitors

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1		Khaled M. Aboshanab	+ 8538 word(s)	8538	2022-03-01 06:54:27	\|
2	deleted the latter paragraphs as the entry length is too long and corrected format.	Beatrix Zheng	-4414 word(s)	4124	2022-03-07 09:49:39	\|

This entry is adapted from the peer-reviewed paper 10.3390/antibiotics11030298

Staphylococcus aureus is a fatal Gram-positive pathogen threatening numerous cases of hospital-admitted patients worldwide. The emerging resistance of the pathogen to several antimicrobial agents has pressurized research to propose new strategies for combating antimicrobial resistance. Novel strategies include targeting the virulence factors of S. aureus. One of the most prominent virulence factors of S. aureus is its eponymous antioxidant pigment staphyloxanthin (STX), which is an auspicious target for anti-virulence therapy.

anti-virulence staphyloxanthin Staphylococcus aureus CrtM CrtN MRSA

1. Naturally Occurring STX Inhibitors

1.1. Flavonoids

Flavone, found in many plants and the mainstay of all flavonoids, is well known to have a wide array of biological activities, such as potent antioxidants, antifbacterial, antifugal as well as anticancer activities. Minimal concentrations, as low as 50 μg/mL, of flavone were found to significantly inhibit the yellow carotenoid pigment in S. aureus. Antibiotic-resistant S. aureus were markedly affected in the presence of flavone which offered a remarkable vulnerability of the strains to hydrogen peroxide. Flavone has also contributed to the inhibition of α-hemolysin and downregulated α-hemolysin gene (hla), resulting in demolishing the hemolysis of red blood cells by S. aureus ^[1]. In 2017, myricetin, a flavonoid commonly present in fruits and vegetables, was reported to inhibit multiple virulence factors in S. aureus. Among the most prominently inhibited virulence factors were staphyloxanthin (STX) pigment production, biofilm formation, adhesion to target cells and red blood cells hemolysis. Gene expression analysis revealed a downregulation of saeR regulator. In vivo, myricetin could efficiently demolish S. aureus infection in a larvae model of Galleria mellonella ^[2]. Although most flavonoids are considered safe, there have been reports of toxic flavonoid–drug interactions, liver failure, contact dermatitis and hemolytic anemia ^[3].

1.2. Rhodomyrtone

A study conducted by Leejae et al. revealed rhodomyrtone, a compound that naturally exists in Rhodomyrtus tomentosa leaves. It is a potent antibacterial agent against many Gram-positive bacteria. It has the advantage of being nontoxic, as its MIC against most of the Gram-positive bacteria is 0.5–1 µg/mL when tested in humans ^[4]. Rhodomyrtone showed promising results when used on S. aureus infected blood specimens in ex vivo assays. Several concentrations of the phytocompound (0.25×, 0.5×, 1×, 2×, and 4× minimum inhibitory concentration (MIC)) were tested on S. aureus isolates. The rhodomyrtone-treated cells were defenseless against the action of innate immunity. A comparative study was held to differentiate between the extent of pigment production in treated and untreated cells with rhodomyrtone. The latter was less prone to oxidative radicals generated by human innate immunity. The action of rhodomyrtone is depicted to be due to its ability to reduce pigment production, leading to an increased susceptibility of the cells to toxic free radicals. Additional effects of rhodomyrtone on bacterial virulence include its effect on the synthesis of DnaK protein, responsible for numerous biological activities of the cells, and/or Sigma factor σB where it has led to the downregulation of this gene resulting in a notable susceptibility of the treated cells to oxidative stress ^[5]. Molecular docking analysis showing 2D (on the right panel) and 3D (on the left panel) representation of interaction patterns of rhodomyrtone with dehydrosqualene synthase receptor is displayed in Figure 1.

Figure 1. Molecular docking analysis showing (two-dimensional) 2D and (three-dimensional) 3D representation of interaction patterns of rhodomyrtone with CrtM receptor ^[6].

1.3. Marine Bioresource: Chitosan

Chitosan is a sugar that is naturally present in the outermost shells of Portunus sanguinolentus. It was found to exhibit several activities such as antibacterial, antifungal and antioxidant activities as well as some anti-virulent characteristics against MRSA. Staphyloxanthin inhibition was among its principal anti-virulent properties when the extracted (100 µg/mL) and commercial (200 µg/mL) forms of chitosan were tested on MRSA strains at their biofilm inhibitory concentrations (BIC). Moreover, the extracted chitosan was found to have a dose-dependent antibiofilm efficacy against various MRSA strains, as well as proved efficacy in disrupting the thick exopolysaccharide (EPS) layer ^[7]. Additionally, chitosan is generally safe and did not have any cytotoxic effects when tested on lung epithelial cell lines ^[8].

1.4. Pogostemon heyneanus and Cinnamomum tamala Essential Oils

Essential oils (EOs) extracted from aromatic plants, such as Pogostemon heyneanus and Cinnamomum tamala, have been tested against MRSA isolates. The EOs were found to exhibit an inhibitory effect on STX production and hemolysin activity at their minimum biofilm inhibitory concentration (MBIC). The essential oils of P. heyneanus and C. tamala showed MIC at the range of (2–6% v/v). The effect of EOs and nerolidol to disrupt the biofilms of MRSA strains was determined at its sub MIC (1/2 and 1/4). P. heyneanus EO showed percentage inhibition of biofilms in the range of 60–80% at concentrations ranging from 3 to 0.5% v/v whereas C. tamala EO showed reduction in biofilms in the range of 55–65% at sub MIC (1–3 v/v). Molecular docking studies were held using CrtM enzyme, the lead enzyme of STX biosynthesis, and the major compounds of EOs. The results revealed that one of the components of EOs, (E)-nerolidol, had higher binding affinity (−6.65 kcal mol⁻¹) to CrtM than other involved compounds ^[9]. A previous study was conducted to evaluate the effectiveness of a mixture of Curcuma longa, Zanthoxylum limonella and Pogostemon heyneanus essential oils in 1:1:2 ratio at 5%, 10% and 20% concentration against blackflies and its dermal toxicity using rat models. The results revealed that the tested repellant was safe and no significant clinical and behavioral changes were detected ^[10].

1.5. 2-Hydroxy-4-Methoxybenzaldehyde (HMB)

Kannappan et al. conducted a study using 2-hydroxy-4-methoxybenzaldehyde (HMB), which was found to confer anti-virulence effects against the notorious MRSA. The MIC of HMB was detected to be 1024 μg/mL. The compound possessed a notable STX inhibition when tested on MRSA at a sub-inhibitory concentration (200 μg/mL). Real time PCR (qPCR) analysis revealed that HMB treatment selectively attenuates stress regulatory genes, such as saeS and SigB in MRSA isolates; the selective effect against those genes results in significant inhibition of the virulence array, such as lipase, hemolysin, and nuclease in MRSA. Furthermore, in vitro studies supported the non-toxic influence of HMB on peripheral blood mononuclear cells (PBMC). As a result, HMB is recognized as a propitious anti-virulence candidate against MRSA ^[11]. Moreover, it was found to have antibacterial, antifungal, antipyretic and antioxidant properties ^[12]. A previous study was carried out on the safety profile of HMB to determine the possibility of being added in and on food as a new flavoring agent. The results revealed that HMB did not have any safety concern when used in the intended levels ^[13].

1.6. Myrtenol

Myrtenol is a well-known phytocompound famous for its agreeable aroma and traditionally known for treating inflammation, anxiety and gastrointestinal pain. It is also considered as a promising herbal product for treatment of allergic asthma ^[14]. It was shown to be successful at inhibiting STX production and biofilm formation in MRSA-treated cells. Its anti-virulence activity has been reported to be concentration-dependent, between 75, 150, and 300 μg ml^–1, without any cytotoxic effect on PBMC. The inhibition of STX production led to increasing the susceptibility of MRSA cells to oxidative radicals as well as human whole blood killing. Downregulation of the global regulator sarA and its mediated virulence genes was revealed upon MRSA cell treatment with myrtenol ^[15].

1.7. Euphorbia tirucalli Latex

Antipathogenic properties of Euphorbia tirucalli latex have been evaluated in vitro and in vivo. The latex did not have an inhibitory effect on the growth of S. aureus. Nonetheless, the latex of Euphorbia tirucalli (LET) showed a remarkable STX reduction ability. In vivo, a LET dose of 10 μL/kg could aid the survival of Tenebrio molitor larvae previously infected with a lethal dose of S. aureus. Hence, LET could set a propitious example of a successful antimicrobial against S. aureus ^[16]. It also has various medicinal uses such as in cases of cough, asthma, warts and rheumatism. The sap of the Euphorbia tirucalli was proved to be irritating to the human eyes and may result in kerato-conjunctivitis ^[17].

1.8. Schinus terebinthifolia Leaf Lectin

In 2019, Lima et al. studied the antimicrobial activity of the lectin derived from the leaves of Schinus terebinthifolia (SteLL) againt S. aureus cells. SteLL treatment succeeded in decreasing STX production in S. aureus using sub-inhibitory concentrations (0.0065× MIC, 0.125× MIC, 0.25× MIC and 0.5× MIC). Furthermore, SteLL synergized ciprofloxacin activity against S. aureus and hence SteLL has been recommended as a novel candidate for anti-virulence therapy ^[18]. Moreover, the result of Ramos et al. 2019 revealed the antitumor activity of SteLL against sarcoma 180. However, hepatic and renal toxicity was observed in animal models ^[19].

1.9. Callistemon citrinus Skeels

Callistemon citrinus Skeels and its isolated compounds are famous for their recognizable pharmaceutical uses such as in rheumatism, diarrhea and dysentery ^[20]. Its antimicrobial activity has been tested on MRSA as well as methicillin-sensitive S. aureus (MSSA) cells to study their anti-virulence abilities. Pulverulentone A (C1), one of the most biologically active isolates of the methylene chloride-methanol extract (MME) of C. citrinus, displayed potent inhibition of STX biosynthesis at half MIC (62.5 μg/mL) in MRSA and MSSA by 55.6% and 54.5%, respectively. It also revealed significant inhibition of biofilm formation at half MIC for up to 71% in MRSA and 62.3% in MSSA ^[21].

1.10. The Essential Oil of Eugenia brejoensis L. (Myrtaceae)

The essential oil of Eugenia brejoensis L. (Myrtaceae) (EbEO) has been studied in Brazil due its antimicrobial activities. It has been found to have a moderate larvicidal activity against the yellow fever mosquito Aedes aegypti ^[22]. The inhibitory profile of EbEO was scrutinized by Filho et al. against various S. aureus isolates including multidrug-resistant (MDR) strains. The essential oil was capable of inhibiting the growth of S. aureus cells at concentrations ranging from 8–516 μg/mL. Using sub-inhibitory concentrations of EbEO on S. aureus, a significant reduction in the production of the carotenoid pigment was observed that led to an impaired defense against oxidative radicals. Furthermore, it reduced the hemolytic activity of S. aureus, thus interfering with its ability to survive in human blood. In vivo studies on Caenorhabditis elegans and G. mellonella infected with S. aureus further confirmed the ability of EbEo treatment to markedly decrease the bacterial load and infection severity in the models ^[23].

1.11. Ginkgo biloba Exocarp Extract

Ginkgo biloba L. exocarp extract (GBEE) is known for having antiproliferative activity on cancer cell lines, with no observable adverse reactions when used in clinical practice ^[24]. Moreover, it is a promising safe approach for treating different neurological disorders such as Alzheimer’s disease, stroke and traumatic brain injury ^[25]. Recent studies have disclosed GBEE’s ability to possess antibacterial effects. Wang et al. examined the inhibitory activities of GBEE on MRSA virulence. The study pointed out that GBEE could inhibit S. aureus and MRSA biofilm synthesis in a concentration-dependent manner. GBEE could also destroy preformed biofilm of the strains at a concentration of 12 μg/mL. Quantitative transcriptional analysis unveiled the downregulation of virulence genes related to resistance as the global regulator gene SigB after 12 h of GBEE treatment, and notable downregulation of icaA and sarA after 6 h of treatment. Furthermore, GBEE resulted in the downregulation of hld gene along with evident STX inhibition ^[26].

1.12. Carvacrol

Carvacrol is a phyto-deravative essential oil well known for its aromatic properties that contribute to its wide use as a spice. It has a wide range of biological activities such as anti-inflammatory, analgesic, antipyretic and antimicrobial activities ^[27]. Moreover, it was found to be clinically safe and tolerable when given to healthy individuals ^[28]. Selvaraj et al. studied the anti-virulence capabilities of carvacrol against MRSA. Sub-inhibitory concentrations of carvacrol (25, 50, and 75 μg/mL) have been tested against numerous MRSA isolates to detect antibiofilm activity. Carvacrol inhibited the production of STX that rendered MRSA isolates susceptible to reactive oxygen species and whole blood killing. Transcriptomic analysis using (qPCR) revealed the downregulation of sarA gene alongside the downregulation of CrtM gene in the treated isolates. Computational molecular docking unveiled a high affinity of carvacrol towards CrtM gene and SarA regulator in S. aureus. In vivo studies hypothesized the capability of carvacrol to mitigate the propagation of MRSA infection in the model as well as maintain a nontoxic effect on the cells of G. mellonella larvae ^[6]. Molecular docking analysis showing two-dimensional (2D) and three-dimensional (3D) representation of interaction patterns of carvacrol with CrtM receptor is displayed in Figure 2.

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Figure 2. Molecular docking analysis showing 2D and 3D representation of interaction patterns of carvacrol with CrtM receptor ^[6].

1.13. Thymol

Thymol has a wide range of therapeutic actions including malignant, cardiovascular and metabolic disorders ^[29]. Valliammai et al. evaluated the inhibitory activity of thymol against the golden-yellow pigment of MRSA strains. Multiple concentrations of thymol (25, 50 and 100 µg/mL) were used to treat MRSA isolates. Dose-dependent STX inhibitory activity of thymol treatment was observed when compared to positive controls with 90% STX inhibition at 100 µg/mL. Growth curve and cytotoxicity analyses revealed the non-killing nature of thymol at the concentration of 100 µg/mL on MRSA cells and its non-cytotoxic effect on PBMC, respectively. In silico and spectrometric analyses suggested that thymol anti-STX activity could be contributed to by its binding affinity to CrtM receptor in MRSA (Figure 3). Furthermore, oxygen susceptibility assays and ex vivo blood survival assay revealed the compromised ability of the treated MRSA cells to resist oxidative radicals. In addition, STX biosynthesis inhibition resulted in enhancing membrane fluidity and sensitized the treated cells to polymyxin B antibiotic ^[30].

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Figure 3. Molecular docking analysis showing 2D (on the right panel) and 3D (on the left panel) representation of interaction patterns of thymol with dehydrosqualene synthase receptor ^[30].

1.14. Hesperidin

Hesperidin is well known in the treatment of type II diabetes and cardiovascular disorders, and has potent anti-inflammatory and antioxidant properties. It is generally safe with no adverse effects when tested on humans and animal models ^[31]. A recent study held by Vijayakumar et al. evaluated the potential anti-infective effect of hesperidin, a flavanone glycoside of plant origin, against MRSA clinical isolates. Hesperidin drastically reduced the production of the carotenoid pigment of MRSA isolates which in turn compromised the survival of MRSA under oxidative stress imposed by H₂O₂. Staphyloxanthin inhibition is believed to be due to the downregulation of crtM gene in MRSA cells. Computational analysis using molecular docking confirmed the capability of hesperidin binding to CrtM proteins involved in STX production (Figure 4). Moreover, hesperidin was proved to have other anti-virulence activities against clinical isolates of S. aureus, such as the significant inhibition of hemolysin, autolysin, and lipase, as well as its antibiofilm potential ^[32].

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Figure 4. Molecular docking analysis showing 3D representation of interaction patterns of hesperidin with dehydrosqualene synthase receptor.

2. Chemically Synthesized Inhibitors

2.1. Indole and Halogenated Indoles

Indole acts as an efficient probe that may help in the development of new drugs treating challenging diseases such as lung cancer. Several studies have emphasized the efficacy of indole derivatives such as indole-3-carbinol and indole-3-carboxaldeh as anti-cancer agents ^[33]. Probing into the effect of indole and its derivatives, one study carried out in vitro and in vivo studies to examine the inhibitory effect of indole and its derivative 7-benzyloxyindole (7BOI) on S. aureus in relevance to STX production and hemolytic activity. S. aureus was more prone to whole blood killing and oxidative stress when treated with indole or its derivative (7BOI). An in vivo model of nematode Caenorhabditis elegans infected with S. aureus and treated with (7BOI) proved successful diminishing of virulence in S. aureus. Real-time qRT-PCR analysis confirmed the downregulation of virulence genes α-hemolysin gene (hla), enterotoxin (seb), and the protease genes (splA), and (sspA) ^[34]. Multiple halogenated indoles were tested against S. aureus to test for potential anti-virulence effects. The 5-iodoindole compound displayed greater potency compared to indole and could efficiently inhibit biofilm formation as well as carotenoid pigment production by S. aureus at 0.3 mM (1/10 of MIC) ^[35].

2.2. Tetrangomycin Derivatives

Tetrangomycin was found to exhibit a significant cytotoxic effect on different cancerous cell lines as well as being a potent free radical scavenger ^[36]. Tetrangomycin derivatives have shown promising anti-virulence activity against S. aureus through STX biosynthesis inhibition. Ribeiro et al. conducted a study to test for the inhibitory characteristics of 27 tetrangomycin derivatives against S. aureus carotenoid pigment. The findings unveiled two of the most potent inhibitory compounds, naphthoquinone dehydro-α-lapachone and 2-Isopropylnaphtho[2,3-b] furan-4,9-dione, and revealed the importance of the presence of lipophilic and hydrogen acceptor moieties around the naphthoquinone ring to achieve STX inhibition. Spectrophotometric analysis was carried out as a simple means for examining the derivative’s mechanism of action on the STX biosynthetic pathway ^[37].

2.3. Repurposing FDA-Approved Drugs

2.3.1. Cholesterol-Lowering Agents

In accordance with Oldfield, the catalysis of two farnesyl diphosphate (FPP) molecules into presqualene diphosphate by CrtM is considered the first basic step in the biosynthesis of staphyloxanthin by S. aureus. The resemblance in structure between CrtM and human squalene synthase (SQS), responsible for cholesterol biosynthesis in humans, aided the repurposing of some cholesterol-lowering agents into STX blockers (Figure 5). The utilization of cholesterol inhibitors as anti-virulence drugs has caused a significant inhibition to S. aureus virulence through directly inhibiting the carotenoid pigment production, rendering the treated strains vulnerable to the oxidative stress of human innate immunity and hence rapid clearance of the microorganism ^[38].

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Figure 5. (A) Staphyloxanthin biosynthesis pathway in S. aureus. (B) Cholesterol and ergosterol biosynthesis pathway.

In 2008, a study conducted by Liu et al. suggested evident structural resemblance between S. aureus CrtM and human SQS. In attribution to the deduction, the analogy between the biosynthetic pathway of cholesterol in humans and that of STX production in S. aureus could be further studied. One cholesterol-lowering agent has been previously tested as a successful STX inhibitor that rendered S. aureus susceptible to oxidative stress of the human neutrophils in a mouse model after depigmenting the strain ^[39]. Later in 2009, Song et al. evaluated the possibility of inhibiting CrtM by potent phosphonosulfonates especially with halogen substitution and were able to prove their inhibitory effect on STX production with no effect against human squalene synthase ^[40].

Similarly, phosphonoacetamides were tested for STX inhibition in vitro and in a mouse infection model where a significant inhibition of disease progression was evident in the latter. X-ray crystallography revealed the most active compound to be N-3-(3-phenoxyphenyl) propylphosphonoacetamide ^[41].

With focus on cholesterol-lowering drugs, lapaquistat acetate and squalestatins are reported to inhibit SQS in humans and hence their cholesterol-lowering activity. Molecular docking analysis was performed to detect the mode of binding of lapaquistat acetate and squalestatin analogs to CrtM enzyme of S. aureus (Figure 6). Molecular docking confirmed the involvement of specific target sites on the CrtM enzyme when introduced to the respective SQS inhibitors. Among the most prominent target residues were His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 ^[42].

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Figure 6. Molecular docking analysis showing 2D (on the right panel) and 3D (on the left panel) representation of interaction patterns of lapaquistat acetate with dehydrosqualene synthase receptor ^[6].

2.3.2. Glyceryl Trinitrate (GTN)

GTN is a well-known medication for the treatment of cardiovascular diseases. Not only does GTN provide renowned anti-angina effects, but in a recent study it was also evident that this drug is capable of STX inhibition, biofilm disruption and oxidative stress resistance in S. aureus strains. Regarding in silico studies, it is reported that GTN binds with high affinity to CrtM which explains its marked STX inhibition activity. Thus, GTN could be a promising antipathogenic candidate against S. aureus ^[43].

2.3.3. Diclofenac

Abbas et al. proposed that the renowned anti-inflammatory drug diclofenac possesses a notable anti-virulence effect against MRSA strains. The assumption was based on the discovery of the drug antipathogenic activity against Pseudomonas aeruginosa and Proteus mirabilis. In this work, diclofenac exerted an anti-STX production activity against MRSA clinical isolates at sub-MICs that reached 8–57.2% when compared to controls. Diclofenac treatment resulted also in decreased biofilm formation (22.67–70%) and noteworthy inhibition of hemolysin activity (5.4–66.34%). The phenotypic results were further confirmed by transcriptomic analysis using quantitative real time PCR that revealed marked downregulation of the previously tested virulence genes. Hence, diclofenac therapy along with other antimicrobials is recommended as an anti-virulence treatment against deleterious MRSA strains ^[44].

2.3.4. Domperidone

Domperidone, an FDA-approved antiemetic drug, was studied by El-Ganiny et al. to detect its potential anti-virulence activity against S. aureus. Significant inhibition of the carotenoid pigment of S. aureus was detected using sub-inhibitory concentrations of domperidone to reach 76.4–81.23% at 1/8 MIC (9.8 μg/mL) and 1/4 MIC (19.5 μg/mL), respectively. Furthermore, the inhibition of the biofilm formation using sub-inhibitory concentrations of domperidone reached 84.37% at 1/4 MIC and 80.16% at 1/8 MIC. Gene expression analysis using qRT-PCR further confirmed the phenotypic results revealing decreased expression levels of virulence genes such as CrtM, SigB, SarA, AgrA, hla, fnbA, and icaA by domperidone treatment ^[45].

2.3.5. Candesartan

Candesartan, a widely used drug in the treatment of high blood pressure, is now being re-studied for inherent anti-virulence characteristics against S. aureus. Candesartan’s ability to inhibit the antioxidant carotenoid pigment of S. aureus was evaluated using sub-inhibitory concentrations of the drug to yield pigment inhibition of 85.57% at 1/4 MIC (1.2 μg/mL) and 80.57% at 1/8 MIC (0.6 μg/mL), respectively. Furthermore, the inhibition of the biofilm formation using sub-inhibitory concentrations of domperidone reached 87.63% at 1/4 MIC and 71.5% at 1/8 MIC. Quantitative gene analysis revealed downregulation of virulence genes of S. aureus with the greatest inhibition activity against CrtM, sigB, sarA, agrA, hla and icaA genes ^[45].

2.3.6. Antifungal Agents

Feifei et al. reported that the antifungal naftifine exerted a potent STX inhibitory activity via competitive inhibition of CrtN enzyme when tested on MSSA cells. The drug was capable of inhibiting the carotenoid pigment without affecting the growth of MSSA cells in a dose-dependent manner (up to 0.2 mM ~64.8 μg/mL) ^[46]. Later in 2020, Jing et al. proposed the synergistic role of naftifine to photodynamic antimicrobial chemotherapy (PACT) against S. aureus. The aiding role of naftifine is believed to be due to its inhibitory activity to STX that scavenges the reactive oxygen species (ROS) generated by PACT. Hence, the notorious antifungal resulted in an enhanced PACT activity when incubated with S. aureus cells at a concentration of 10 μM ^[47].

A recent study carried by El-Ganiny et al. focused on miconazole, which was reported to exhibit anti-virulence effects when studied against S. aureus standard strain (well-characterized strain with defined susceptibility or resistance profiles to the antimicrobial agents tested). In that study, 1/4 MIC (18.75 μg/mL) and 1/8 MIC (9.4 μg/mL) of miconazole were used to inhibit multiple virulence characteristics of S. aureus. STX inhibition reached 76.43–83.93% upon treatment with the indicated sub-inhibitory concentrations of the drug. Furthermore, the inhibition of the biofilm formation using sub-inhibitory concentrations of domperidone reached 90% at 1/4 MIC and 86.84% at 1/8 MIC. Transcriptomic analysis using qT-PCR divulged the reduced expression of CrtM, SigB, SarA, AgrA, hla, FnbA, and IcaA ^[45].

2.4. Newly Discovered CrtN Inhibitors

2.4.1. 5 m Analog

Wang et al. have previously revealed that CrtN is a promising target for anti-virulence therapy. They have further disclosed the ability of the famous antifungal naftifine to drastically abolish the carotenoid pigment production in S. aureus species. The discovery of 5 m, a novel type of Benzofuran-derived CrtN inhibitor, has recently followed in the footsteps of the repurposing strategy of naftifine. As a result, the analogy has reflected a typical effect of the 5 m analog on S. aureus Newman and three other methicillin-resistant strains with low IC50 values ranging from 0.38–5.45 nM. The treated cells were rendered susceptible to immune clearance and their virulence was markedly weakened ^[48].

2.4.2. Compound NP16

A newly discovered compound termed NP16 has showed a potent activity as a CrtN inhibitor in S. aureus strains. Consequently, notable interruption to the golden carotenoid pigment biosynthesis was evident that further caused an increased vulnerability to oxidative stress and neutrophil killing in vivo ^[49].

2.4.3. 1,4-Benzodioxan-derivatives

In 2018, 38 1,4-benzodioxan-derived CrtN inhibitors were synthesized to combat the downsides of the leading compound 4a. Derivative 47 exhibited a remarkable CrtN inhibitory effect with higher potency than 4a (pigment inhibition in S. aureus Newman: IC50 = 270.4 ± 43.8 nM by compound 47 vs. IC50 = 1.9 nM by compound 4a) in addition to enhanced water solubility. The sensitization effect of derivative 47 on MRSA strains was reported to be quite significant and successfully facilitated immune clearance in vitro ^[50].

2.5. Others

Farnesol

Candida albicans and S. aureus are among the most commonly known opportunistic pathogens that usually co-exist in mixed biofilms ^[51]. Both pathogens are often isolated together from hospital-admitted patients ^[52]. In a recent study, the role of C. albicans-secreted quorum sensing (QS) molecule (farnesol) was assessed against S. aureus cells. The study mimicked a mixed biofilm of C. albicans with S. aureus cells by repetitive exposure of S. aureus to farnesol. The sensitized S. aureus cells revealed significant inhibition of STX. The findings of transcriptional analysis further displayed marked changes in the expression of global regulators involved in resistance to oxidative stress. Unfortunately, the activation of stress-response mechanisms in S. aureus boosted its tolerance to intracellular killing and ROS. The pigment inhibition effect was reasoned then by proposing a theoretical binding model that indicated the binding of farnesol to CrtM enzyme causing blockage of the biosynthetic pathway of STX due to its structural resemblance to the substrate of CrtM. Those findings illustrate the role of the fungal-secreted QS mediator that could successfully elicit oxidative stress on S. aureus through thiol-based redox system activation. Moreover, the results of the previous study reported that depigmentation mediated by STX inhibitors was considered a transient conditional state, as upon the gradual removal of farnesol, gradual recovery of the pigment was observed in comparison with the control cells ^[53].

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