Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes

Hsieh, Ying-Hsin; Zou, Juan; Jin, Jinshan; Yang, Hanfang; Chen, Yanyi; Jiang, Chun; Yang, Jenny J.; Tai, Phang C.

doi:10.1016/j.ab.2015.04.005

Cited by 4 publications

(26 citation statements)

References 43 publications

(86 reference statements)

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“…[3b, 12a] Among the Gram-negative bacteria we tested in the synergistic use of PMBN or NAB7061 and SecA inhibitors (Table 2), many are MDR pathogens. In some tests, RB was used a reference compound, which has been extensively tested [7b] and is the lead compound of Class A RB analogues, [7a, 13] with sub-μM potency in inhibiting SecA ATPase and other functional activities. [7a, 13] Many other inhibitors (Table 2) showed potency in the low μg/mL to high ng/mL MIC range.…”

Section: Resultsmentioning

confidence: 99%

“…In some tests, RB was used a reference compound, which has been extensively tested [7b] and is the lead compound of Class A RB analogues, [7a, 13] with sub-μM potency in inhibiting SecA ATPase and other functional activities. [7a, 13] Many other inhibitors (Table 2) showed potency in the low μg/mL to high ng/mL MIC range. All 3 classes of inhibitors exhibited bactericidal effects to different degrees.…”

Section: Resultsmentioning

confidence: 99%

“…[13] Its smaller analogues such as SCA-50 are more selective. [7a, 13] Kinetic studies using the channel activity of SecA-only liposomes assayed in the oocytes (Figure 3) showed that all 3 classes of inhibitors Class A SCA-50, Class B SCA-15, and Class C SCA-107 ( [27] used here for comparison) are non-competitive against ATP for EcSecA, PaSecA or StSecA (Figure 3). Thus major inhibitory effects in the membrane may not be on the high-affinity ATPase active site directly (Table 5C); rather, the inhibition is probably allosteric in a membrane lipid environment (SFig.…”

Section: Resultsmentioning

confidence: 99%

“…Molecular docking studies using methods as described previously [13, 23a] show that SCA-107 lies outside the ATP pocket of chain A (SFig. 3A).…”

Section: Resultsmentioning

confidence: 99%

“…This is different from the binding site of SCA-50 which blocks the ATP entrance in chain B. [13] Also, docking studies indicate that EcSecA-L515, (a point SecA mutation which results in resistance to SCA-107, see below) is located away (SFig. 3B) from the SCA-107 binding pockets (and possibly SCA-50’s), suggesting potential allosteric effects.…”

Section: Resultsmentioning

confidence: 99%

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Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

et al. 2016

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With the wide-spread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram-negative bacteria. Along this line, the identification of viable targets is a critical first step. SecA is a protein translocase and is commonly believed to be an excellent target for the development of broad-spectrum antimicrobials. In recent years, we have developed three structural classes of SecA inhibitors, which have proven to be very effective against Gram-positive bacteria. However, we have not achieved the same level of success against Gram-negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain understanding as to why these inhibitors were not effective against Gram-negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram-positive and Gram-negative bacteria is in the additional permeability barrier posed by the outer membrane in Gram-negative bacteria. We have also found that expression of efflux pumps, which are responsible for multi-drug resistance, have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor-resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA-azi-9 mutation increased the resistance, providing genetic evidence that SecA indeed is the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram-negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…Molecular docking studies using methods as described previously [13, 23a] show that SCA-107 lies outside the ATP pocket of chain A (SFig. 3A).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

et al. 2016

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Thiouracil SecA inhibitors: bypassing the effects of efflux pumps and attenuating virulence factor secretion in MRSA and Bacillus anthracis

et al. 2021

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We have previously reported a series of thiouracil analogs as bacterial SecA inhibitors. In this study, one potent thiouracil analog, SCA-15, was further evaluated for its inhibitory effects on SecA proteins and against strains of methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus anthracis Sterne. SCA-15 inhibited the ion-channel activities of SecA1 with IC 50 of 2.0-2.8 μM and the ATPase activities of SecA2 with IC 50 of 13-20 μM, respectively. Drug affinity responsive target stability (DARTS) and protein pull-down experiments further supported SaSecA1 and SaSecA2 as the targets of the inhibitor. SCA-15 showed promising bactericidal effects against MRSA and B. anthracis Sterne. In addition, SCA-15 could at least partially overcome the effects of efflux pumps responsible for multidrug resistance. Moreover, SCA-15 also inhibited the secretion of several important toxins of both S. aureus and B. anthracis. These results indicated that targeting SecA is a promising antimicrobial strategy against MRSA and B. anthracis.

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SecA inhibitors as potential antimicrobial agents: differential actions on SecA-only and SecA-SecYEG protein-conducting channels

Jin

Hsieh

Chaudhary

et al. 2018

FEMS Microbiology Letters

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Sec-dependent protein translocation is an essential process in bacteria. SecA is a key component of the translocation machinery and has multiple domains that interact with various ligands. SecA acts as an ATPase motor to drive the precursor protein/peptide through the SecYEG protein translocation channels. As SecA is unique to bacteria and there is no mammalian counterpart, it is an ideal target for the development of new antimicrobials. Several reviews detail the assays for ATPase and protein translocation, as well as the search for SecA inhibitors. Recent studies have shown that, in addition to the SecA-SecYEG translocation channels, there are SecA-only channels in the lipid bilayers, which function independently from the SecYEG machinery. This mini-review focuses on recent advances on the newly developed SecA inhibitors that allow the evaluation of their potential as antimicrobial agents, as well as a fundamental understanding of mechanisms of SecA function(s). These SecA inhibitors abrogate the effects of efflux pumps in both Gram-positive and Gram-negative bacteria. We also discuss recent findings that SecA binds to ribosomes and nascent peptides, which suggest other roles of SecA. A model for the multiple roles of SecA is presented.

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Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes

Cited by 4 publications

References 43 publications

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

Thiouracil SecA inhibitors: bypassing the effects of efflux pumps and attenuating virulence factor secretion in MRSA and Bacillus anthracis

SecA inhibitors as potential antimicrobial agents: differential actions on SecA-only and SecA-SecYEG protein-conducting channels

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