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Article

Identification of Novel Antistaphylococcal Hit Compounds Targeting Sortase A

by
Galyna Volynets
1,2,
Hanna Vyshniakova
3,
Georgiana Nitulescu
4,*,
George Mihai Nitulescu
4,
Anca Ungurianu
4,
Denisa Margina
4,
Olena Moshynets
1,
Volodymyr Bdzhola
1,
Ihor Koleiev
5,
Olga Iungin
1,
Sergiy Tarnavskiy
1 and
Sergiy Yarmoluk
1
1
Institute of Molecular Biology and Genetics, The NAS of Ukraine, 150 Zabolotnogo St., 03143 Kyiv, Ukraine
2
Scientific Services Company Otava Ltd., 150 Zabolotnogo St., 03143 Kyiv, Ukraine
3
L.V. Gromashevsky Institute of Epidemiology and Infectious Diseases NAMS of Ukraine, 5 Amosova St., 03038 Kyiv, Ukraine
4
Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, Traian Vuia 6, 020956 Bucharest, Romania
5
Educational and Scientific Center “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv 64/13, Volodymyrska Str., 01601 Kyiv, Ukraine
*
Author to whom correspondence should be addressed.
Molecules 2021, 26(23), 7095; https://doi.org/10.3390/molecules26237095
Submission received: 3 November 2021 / Revised: 23 November 2021 / Accepted: 23 November 2021 / Published: 24 November 2021
(This article belongs to the Section Medicinal Chemistry)
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Abstract

:
Staphylococcus aureus (S. aureus) is a causative agent of many hospital- and community-acquired infections with the tendency to develop resistance to all known antibiotics. Therefore, the development of novel antistaphylococcal agents is of urgent need. Sortase A is considered a promising molecular target for the development of antistaphylococcal agents. The main aim of this study was to identify novel sortase A inhibitors. In order to find novel antistaphylococcal agents, we performed phenotypic screening of a library containing 15512 compounds against S. aureus ATCC43300. The molecular docking of hits was performed using the DOCK program and 10 compounds were selected for in vitro enzymatic activity inhibition assay. Two inhibitors were identified, N,N-diethyl-N′-(5-nitro-2-(quinazolin-2-yl)phenyl)propane-1,3-diamine (1) and acridin-9-yl-(1H-benzoimidazol-5-yl)-amine (2), which decrease sortase A activity with IC50 values of 160.3 µM and 207.01 µM, respectively. It was found that compounds 1 and 2 possess antibacterial activity toward 29 tested multidrug resistant S. aureus strains with MIC values ranging from 78.12 to 312.5 mg/L. These compounds can be used for further structural optimization and biological research.

1. Introduction

Staphylococcus aureus (S. aureus) is a causative agent of the majority of skin infections, hospital-acquired infections and severe diseases such as bacteremia, sepsis, meningitis, osteomyelitis, endocarditis, and pneumonia [1].
S. aureus belongs to the group of high-priority dangerous “ESKAPE” pathogens, which includes the multidrug resistant isolates of Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas and Enterobacter that are currently considered as the greatest challenge in medicine. Many hospital-acquired S. aureus isolates have become resistant to most available antibiotics. Staphylococcal resistance to penicillin is mediated by penicillinase which hydrolyses the β-lactam ring of antibiotic. In clinical practice for treatment of staphylococcal infections, methicillin, chemically modified penicillin which cannot be hydrolyzed by penicillinase, has been widely used. At the present time, methicillin-resistant S. aureus (MRSA), which is resistant to all of the β-lactam antibiotics due to modification of penicillin-binding protein, has become one of the most dangerous pathogens. The antibiotic of choice in a case of MRSA-associated infection is vancomycin. However, vancomycin-resistant MRSA strains have emerged recently [2]. It should be noted that no other antibiotic to date has shown any superiority to vancomycin in the treatment of MRSA infections with the possible exception of linezolid in hospital-acquired pneumonia [3]. Therefore, the development of multidrug resistance in S. aureus has caused an urgent need for the search of novel effective targets and corresponding inhibitors to develop principally new antibiotics effective against strains which are resistant to already known antibiotics of choice.
Sortase A is considered as a promising molecular target for the development of antistaphylococcal agents. Sortase A is a membrane-bound transpeptidase which catalyzes the transfer and immobilization of essential virulence factors to the surface of microorganisms. Inhibitors of sortase A affect virulence and biofilm formation, therefore decreasing selective pressure which can cause the development of antibiotic resistance [4]. Sortase A is not presented in eukaryotic organisms, hence the inhibitors of this enzyme may possibly have less toxicity for human organisms. Since this enzyme is membrane-bound, the inhibitors do not have to penetrate into the cell.
To date, small-molecular inhibitors of sortase A have been reported among the derivatives of diarylacrylonitrile [5,6], aryl(β-amino)ethyl ketone [7], rhodanine, pyridazinone, pyrazolethione [8], morpholinobenzoate, aryl 3-acryloamides [9,10], dihydro-β-carboline [11], benzo[d]isothiazol-3(2H)-one-adamantane amine [12], 3,6-disubstituted triazolothiazole [13], 2-phenylbenzofuran-3-carboxamide [14], 2-phenylbenzo[d]oxazole-7-carboxamide [15], 2-(2-phenylhydrazinylidene)alkanoic acid [16], indolethiazolidine [17], pyrrolomycin [18], 2-phenylthiazole [19], 2,5-disubstituted thiadiazole [20], thiadiazolinedione [21], disulfanylbenzamide [22], and 1,2,4-oxadiazole topsentin analogs [23]. Furthermore, several inhibitors for sortase A have been identified among natural products such as β-sitosterol-3-O-glucopyranoside [24], berberine chloride [25], bis(indole)-alkaloid [26], isoaaptamine [27], flavonoids (kurarinol [28], myricetin [29], quercitrin [30], morin [31], eriodictyol [32], acacetin [33], 7-hydroxy-6-methoxy-flavanone and formononetin [34], dryocrassin ABBA [35]), curcumin [36], maltol-3-O-(4′-O-cis-p-cumaroyl-6′-O-(3-hydroxy-3-methylglutaroyl)-β-glucopyranoside [37], skyrin [38], aspermytin A [39], natural naphthoquinones [40], isovitexin [41], coumarines [42], taxifolin [43], erianin [44], quinone [45], chalcone [46], bis(indole) alkaloids [47], orientin [48], gallotannins [49] and peptides from the marine-derived fungi Aspergillus allahabadii [50]. Several review works describe important small organic compounds that act as potent sortase A inhibitors [51,52,53,54]. In the present article, we report two novel inhibitors of S. aureus sortase A belonging to novel chemical classes, the derivatives of acridin-9-yl-(1H-benzoimidazol-5-yl)-amine and 2-phenyl-quinazoline, which possess antimicrobial activity toward multidrug resistant S. aureus strains.

2. Results

In order to find novel antistaphylococcal agents, phenotypic screening of a library containing 15,512 compounds, provided by commercial supplier OTAVA Ltd., was performed by the Community for Open Antimicrobial Drug Discovery (CO-ADD), against methicillin-resistant S aureus strain ATCC43300. As a result, 250 compounds inhibiting growth of MRSA ATCC43300 at least by 30% at the concentration of 32 mg/L were found. The list of active compounds in SMILES format is available in the Supplementary Materials. To identify which compounds can potentially inhibit S. aureus sortase A, we performed molecular docking of 250 compounds into the active site of this enzyme using the DOCK program. According to the results of molecular docking calculations and visual inspection of the best-scored complexes, we selected 10 compounds for in vitro testing. The chemical structure of compounds and their antibacterial activity toward Staphylococcus aureus are presented in Table 1.
The tested compounds inhibited the growth of the methicillin-resistant S. aureus strain ATCC43300 with inhibition percentages ranging from 38.98 up to 99.57%. Compounds 2 and 4 almost completely inhibited the bacterial growth after exposure at 32 mg/L and could be used to develop new antibacterial substances.

2.1. Sortase A Activity Assay

Among these ten investigated compounds, using in vitro sortase A activity assay we found two inhibitors of S. aureus sortase A, N,N-diethyl-N′-(5-nitro-2-(quinazolin-2-yl)phenyl)propane-1,3-diamine (1) and acridin-9-yl-(1H-benzoimidazol-5-yl)-amine (2), which decreased enzyme activity with IC50 values of 160.3 µM and 207.01 µM, respectively (Figure 1). The other eight compounds produced only a small inhibition. Therefore, these compounds possess other molecular mechanisms of action. Possibly, compound 4, with high antibacterial activity, should be investigated for inhibitory activity toward S. aureus DNA gyrase, since according to recent literature data, several indole derivatives demonstrate antistaphylococcal activity targeting this enzyme [55].

2.2. Molecular Docking

According to molecular docking results, the compound N,N-diethyl-N′-(5-nitro-2-(quinazolin-2-yl)phenyl)propane-1,3-diamine (1) is involved in hydrophobic interactions with amino acid residues Val166 and Ile182 in the active site of sortase A. Furthermore, the nitrogen atom of the quinazolin ring forms a hydrogen bond with conserved amino acid residue Arg197, which is important for catalysis and belongs to catalytic triad (His120, Cys184, and Arg197). Quinazolin heterocycle is also implicated in π-cation interaction with Arg197 (Figure 2).
The inhibitor acridin-9-yl-(1H-benzoimidazol-5-yl)-amine is involved in hydrophobic interactions with amino acid residues Ile199, Ile182, Cys184, Trp194 and benzoimidazol heterocycle forms π-cation interactions with Arg197 (Figure 3).

2.3. Antibacterial Assay

The compounds 1 and 2 were extensively screened for antibacterial activity toward multidrug resistant S. aureus strains isolated in Ukrainian hospitals. The minimum inhibitory concentration (MIC) values for compounds 1 and 2 toward S. aureus isolates are presented in the Table 2.
The MIC values ranged between 78.12 and 312.5 mg/L for compound 1 and in the range of 156.2 and 312.5 mg/L for compound 2. Compound 1 was more active than compound 2 on 12 of the 29 bacterial strains, while compound 2 had a higher antimicrobial effect on 3 strains. This observation is correlated with the higher inhibitory effect of compound 1 on the bacterial sortase.
The bacterial strains were investigated for sensitivity to antibiotics by the disco diffusion method using Mueller–Hinton Agar. The visual analysis of antibiotic sensitivity was performed according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) recommendations [56] and the results are presented in Table 3.

3. Discussion

Using molecular docking techniques, we identified two novel inhibitors of S. aureus sortase A, N,N-diethyl-N′-(5-nitro-2-(quinazolin-2-yl)phenyl)propane-1,3-diamine (1), and acridin-9-yl-(1H-benzoimidazol-5-yl)-amine (2), which possess moderate enzyme inhibitory activity with IC50 values of 160.3 µM and 207.01 µM, respectively. When comparing the binding mode of highly potent benzisothiazolinone-based inhibitor in the crystal structure of S. aureus sortase A (PDB ID: 2MLM) [12], which was used for molecular docking, and docked complexes of compounds 1 (Figure 1) and 2 (Figure 2), it can be concluded that the simultaneous formation of a hydrogen bond with conservative Arg197 and tight hydrophobic interactions with Trp194, which were established in the co-crystal structure, can be important for inhibitory potency. It should be noted that in our study, compounds 1 and 2 form only one from these two intermolecular interactions—compound 1 builds hydrogen bond with Arg197 and compound 2 forms hydrophobic interactions with Trp194. Possibly, further chemical optimization of compounds 1 and 2 should be performed in order to reach both these types of ligand–receptor interactions.
Antibacterial studies revealed that compounds 1 and 2 inhibit growth of a number of tested multidrug resistant S. aureus strains with MIC values in the range from 78.12 to 312.5 mg/L (Table 2). The antibacterial activity of sortase A inhibitors is low in comparison to known antistaphylococcal antibiotics such as vancomycin and linezolid, which have MIC values about 1 mg/L. Taking into account high levels of resistance to standard antibiotics, the development of antibiotics with novel mechanisms of action is of urgent need.
As it can be seen from the Table 2, compound 1 is more profitable than compound 2 and reveals the antimicrobial activity toward S. aureus strains which have different profiles of antibiotic sensitivity with MIC values in the range from 78.12 to 312.5 mg/L, while compound 2 demonstrates antimicrobial activity with MIC values in the range from 156.2 to 312.5 mg/L (Table 3).
Compound 1 possess the highest antibacterial activity with MIC value of 78.12 mg/L toward multidrug resistant S. aureus strains 1012, 501, 502, 504, 506, 510, 511. All these strains have susceptibility to chloramphenicol, moxifloxacin and linezolid. Vice versa, it was revealed that the compound 1 has the lowest antibacterial activity toward S. aureus isolates 964, 997 and 1013 which all have resistance to chloramphenicol; two of them (964, 997) have resistance to moxifloxacin and one of them (997) has resistance to linezolid. Compound 2 has antibacterial activity toward tested S. aureus strains, mostly with a MIC value of 156.2 mg/L, except three isolates, 854, 890 and 892, which all are susceptible to linezolid. Therefore, compounds 1 and 2 possess different effectiveness toward S. aureus strains with various antibiotic resistance profiles and can be useful for further optimization and development of novel lead compounds with antibacterial activity toward multidrug resistant S. aureus strains.

4. Materials and Methods

4.1. Sortase A Activity Assay

The inhibitory activity of compounds was determined by quantifying the fluorescence intensity upon 5-FAM/QXL® substrate cleavage using the SensoLyte® 520 Sortase A Activity Assay Kit (Anaspec, San Jose, CA, USA). The compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted with distilled water until the concentration of DMSO was 1% and the solution’s intrinsic fluorescence was checked. Each compound was tested at 7 concentration levels in the range of 100–0.1 µM. According to kit protocol, the assay was performed in a 96-well plate, each well containing 10 µL test compound solution, 40 µL enzyme solution and 50 µL substrate solution. We used as controls the enzyme, a 1% DMSO solution, the substrate solution, and 4-hydroxymercuribenzoic acid (HMB) as the positive control. The enzymatic assay was performed for 60 min at room temperature and analyzed fluorometrically (SpectraMAX Gemini XS, San Jose, CA, USA) at Ex/Em = 490 nm/520 nm. All reported values are the means of duplicate experiments.

4.2. Molecular Docking

The molecular docking was carried out with DOCK program [57,58,59,60]. As a receptor we used crystal structure of S. aureus sortase A with PDB ID: 2MLM [12]. The geometry of ligands was calculated using YFF force field [61]. The hydrogen atoms were added with Open Babel v 2.4.0 [62]. Partial atomic charges of the ligands were added with Kirchhoff method [63].
Docking parameters were set as described earlier [64] with several modifications. In our experiments, as the active site atoms we selected the atoms of amino acid residues within 10 Å from the reference ligand—benzo[d]isothiazol-3-one. The spheres in the active site for semi-flexible ligand docking were set with DOCK sphgen software. Grid maps were calculated using Grid program, with grid spacing 0.3 Å. Proteins were represented by the all atom model. We used ‘multiple anchors’ parameter for virtual screening, the minimum of heavy atoms in the anchor was set to 6, and the maximum number of orientations was 1000.
Visual inspection of the complexes of compounds with sortase A was performed using Discovery Studio Visualizer 4.0 [65].

4.3. Antibacterial Assay

All bacteria were cultured in Cation-adjusted Mueller Hinton broth (CAMHB) at 37 °C overnight. A sample of each culture was then diluted 40-fold in fresh broth and incubated at 37 °C for 1.5–3 h. The resultant mid-log phase cultures were diluted (CFU/mL measured by OD600), then added to each well of the compound-containing plates, giving a cell density of 5 × 105 CFU/mL and a total volume of 50 μL. All the plates were covered and incubated at 37 °C for 18 h without shaking.
Growth inhibition of all bacteria was determined measuring absorbance at 600 nm (OD600), using a Tecan M1000 Pro monochromator plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references. The growth rates for bacteria had a variation of ±10%, which is within the reported normal distribution of bacterial growth.
The MIC was determined as the lowest concentration at which the growth was fully inhibited, defined by an inhibition ≥80%. In addition, the maximal percentage of growth inhibition is reported as DMax, indicating any compounds with partial activity.

Supplementary Materials

The following are available online, list of active compounds in SMILES format.

Author Contributions

Conceptualization, V.B.; Methodology, G.V., H.V., G.N., G.M.N., A.U., D.M., O.M., V.B., I.K., O.I., S.T. and S.Y.; Supervision, S.Y.; Writing—original draft, G.V., H.V., G.N., G.M.N., A.U., D.M., O.M., V.B., I.K., O.I., S.T. and S.Y.; Writing—review and editing, I.K. and S.Y. All authors have read and agreed to the published version of the manuscript.

Funding

The APC was funded by the authors. Antimicrobial screening toward methicillin-resistant Staphylococcus aureus strain ATCC43300 was performed by CO-ADD (The Community for Open Antimicrobial Drug Discovery), funded by the Wellcome Trust (UK) and The University of Queensland (Australia).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

Antimicrobial screening toward methicillin-resistant Staphylococcus aureus strain ATCC43300 was performed by CO-ADD (The Community for Open Antimicrobial Drug Discovery), funded by the Wellcome Trust (UK) and The University of Queensland (Australia).

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds 1–10 are available from the authors.

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Molecules 26 07095 g001 550
Figure 1. The plotted sortase A inhibition (I%) against compounds logarithm of concentration (μM): (a) compound 1; (b) compound 2.
Figure 1. The plotted sortase A inhibition (I%) against compounds logarithm of concentration (μM): (a) compound 1; (b) compound 2.
Molecules 26 07095 g001
Molecules 26 07095 g002 550
Figure 2. The binding mode of compound 1 in the active site of S. aureus sortase A; hydrogen bond is shown by the green dotted line, hydrophobic interactions are indicated by the magenta dotted lines and π-cation interaction is presented by orange dotted line.
Figure 2. The binding mode of compound 1 in the active site of S. aureus sortase A; hydrogen bond is shown by the green dotted line, hydrophobic interactions are indicated by the magenta dotted lines and π-cation interaction is presented by orange dotted line.
Molecules 26 07095 g002
Molecules 26 07095 g003 550
Figure 3. The binding mode of compound 2 in the active site of S. aureus sortase A; hydrogen bond is shown by the green dotted line, hydrophobic interactions are indicated by the magenta dotted lines and π-cation interactions are presented by the orange dotted lines.
Figure 3. The binding mode of compound 2 in the active site of S. aureus sortase A; hydrogen bond is shown by the green dotted line, hydrophobic interactions are indicated by the magenta dotted lines and π-cation interactions are presented by the orange dotted lines.
Molecules 26 07095 g003
Table
Table 1. Structures and antibacterial activity (percentage growth inhibition) against S. aureus MRSA ATCC43300 for compounds 1–10 at 32 mg/L.
Table 1. Structures and antibacterial activity (percentage growth inhibition) against S. aureus MRSA ATCC43300 for compounds 1–10 at 32 mg/L.
No.StructureConcentration (μM)Antibacterial Activity on S. aureus MRSA ATCC43300 (%)
1 Molecules 26 07095 i0017758.75
2 Molecules 26 07095 i0029299.57
3 Molecules 26 07095 i00311865.25
4 Molecules 26 07095 i0049898.51
5 Molecules 26 07095 i00510351.2
6 Molecules 26 07095 i0066538.98
7 Molecules 26 07095 i0077443.25
8 Molecules 26 07095 i00812989.6
9 Molecules 26 07095 i0099886.65
10 Molecules 26 07095 i0107575.5
Table
Table 2. The antimicrobial activity of compounds 1 and 2 toward multidrug resistant S. aureus strains.
Table 2. The antimicrobial activity of compounds 1 and 2 toward multidrug resistant S. aureus strains.
S. aureus StrainsMIC (mg/L)
Compound 1Compound 2
S. aureus 2921378.12156.2
MR 43378.12156.2
S. aureus 854156.2312.5
S. aureus 887156.2156.2
S. aureus 890156.2312.5
S. aureus 892156.2312.5
S. aureus 938156.2156.2
S. aureus 964312.5156.2
S. aureus 997312.5156.2
S. aureus 101278.12156.2
S. aureus 1013312.5156.2
S. aureus 1021156.2156.2
S. aureus 584156.2156.2
S. aureus 585156.2156.2
S. aureus 586156.2156.2
S. aureus 522156.2156.2
S. aureus 523156.2156.2
S. aureus 524156.2156.2
S. aureus 50178.12156.2
S. aureus 50278.12156.2
S. aureus 503156.2156.2
S. aureus 50478.12156.2
S. aureus 505156.2156.2
S. aureus 50678.12156.2
S. aureus 507156.2156.2
S. aureus 508156.2156.2
S. aureus 509156.2156.2
S. aureus 51078.12156.2
S. aureus 51178.12156.2
Table
Table 3. The sensitivity of S. aureus strains to antibiotics.
Table 3. The sensitivity of S. aureus strains to antibiotics.
StrainPenicillinOxacillinErythromycinClindamycinGentamicinChloramphenicolCiprofloxacinCefazolinAzithromycinVancomycinLinezolidTeicoplaninMoxifloxacin
S. aureus 854RIRIRSIRSSIRIRRSIRS
S. aureus 887RIRSSIRSSIRIRIRSIRS
S. aureus 890RRRRRRIRRRRSIRIR
S. aureus 892RIRSSIRSIRSIRIRSIRS
S. aureus 938RIRSSIRSSIRIRIRSIRS
S. aureus 964RRRRIRIRIRSRIRSIRIR
S. aureus 997RSRRIRIRIRSIRRRRIR
S. aureus 1012SSSSIRSSIRIRRSIRS
S. aureus 1013RSIRIRIRRSSIRIRSIRS
S. aureus 1021RSSSIRIRIRIRIRIRIRIRS
S. aureus 584RIRRSIRSSIRIRIRSSS
S. aureus 585RRIRSRSRIRRIRSIRIR
S. aureus 586RRIRSRRRRIRIRSIRIR
S. aureus 522RRIRIRRRRIRIRIRSIRIR
S. aureus 523RRIRIRRRRIRIRIRSIRIR
S. aureus 524RIRRRIRSIRIRRSSSS
S. aureus 501RRRRIRSIRSRSSIRS
S. aureus 502RRRRIRSIRSIRIRSIRS
S. aureus 503RRRIRRSIRSRRSIRS
S. aureus 504RRIRIRIRSSSRIRSIRS
S. aureus 505RRRRIRSIRSRIRSIRS
S. aureus 506RRRIRIRSIRSRIRSSS
S. aureus 507RRRRIRSIRSRSSSS
S. aureus 508RRRIRIRSIRSRIRSIRS
S. aureus 509RRRSRSSSRIRSIRS
S. aureus 510RRRIRIRSSIRRIRSIRS
S. aureus 511RIRRSRSIRIRRIRSIRS
R—resistance; IR—intermediate resistance; S—susceptibility.
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Volynets, G.; Vyshniakova, H.; Nitulescu, G.; Nitulescu, G.M.; Ungurianu, A.; Margina, D.; Moshynets, O.; Bdzhola, V.; Koleiev, I.; Iungin, O.; et al. Identification of Novel Antistaphylococcal Hit Compounds Targeting Sortase A. Molecules 2021, 26, 7095. https://doi.org/10.3390/molecules26237095

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Volynets G, Vyshniakova H, Nitulescu G, Nitulescu GM, Ungurianu A, Margina D, Moshynets O, Bdzhola V, Koleiev I, Iungin O, et al. Identification of Novel Antistaphylococcal Hit Compounds Targeting Sortase A. Molecules. 2021; 26(23):7095. https://doi.org/10.3390/molecules26237095

Chicago/Turabian Style

Volynets, Galyna, Hanna Vyshniakova, Georgiana Nitulescu, George Mihai Nitulescu, Anca Ungurianu, Denisa Margina, Olena Moshynets, Volodymyr Bdzhola, Ihor Koleiev, Olga Iungin, and et al. 2021. "Identification of Novel Antistaphylococcal Hit Compounds Targeting Sortase A" Molecules 26, no. 23: 7095. https://doi.org/10.3390/molecules26237095

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