In Silico Structure-Based Screening of Large Ligand Library Against Virulence Factors of Drug Resistant Pathogens

The discovery and continual development of antibiotics since the 1940s have greatly reduced the mortality rate of infectious diseases. However, newly emerging and reemerging of drug-resistant microbes mean that despite our best efforts, infectious diseases remain a great threat to humanity today [1]. Among these, Staphylococcus aureus and Mycobacterium tuberculosis (MTB) are especially problematic. S. aureus is a highly adaptive gram-positive bacterium and commensal of the human skin and nostrils [2]. S. aureus is a common cause of minor skin and wound infections, but can also cause serious and even fatal infections, particularly in the immunocompromised persons. Methicillin-resistant Staphylococcus aureus (MRSA) infection is becoming an alarming local Received: June 03, 2019


Introduction
The discovery and continual development of antibiotics since the 1940s have greatly reduced the mortality rate of infectious diseases. However, newly emerging and reemerging of drug-resistant microbes mean that despite our best efforts, infectious diseases remain a great threat to humanity today [1]. Among these, Staphylococcus aureus and Mycobacterium tuberculosis (MTB) are especially problematic. S. aureus is a highly adaptive gram-positive bacterium and commensal of the human skin and nostrils [2].
S. aureus is a common cause of minor skin and wound infections, but can also cause serious and even fatal infections, particularly in the immunocompromised persons. Methicillin-resistant Staphylococcus aureus (MRSA) infection is becoming an alarming local The emergence of drug-resistant microbes is an alarming threat to the global population. There is an urgent need of effective drugs against these pathogens. Among these, SA and MTB are especially problematic to human. The global spread of MRSA is of great concern in the treatment of Staphylococcal infections because it is quickly acquiring resistance to all clinical antibacterial agents. A novel approach of drug development is to target virulence factors, which can potentially prevent drug-resistance from building up. An early secretory SA protein EsxA is known to be a virulence factor which plays a major role in the pathogenesis of the bacterium. Therefore, EsxA is a promising drug target. An in-silico platform has been setup to perform structure-based screening of a large compound library containing 6.8 million lead-like and bioactive ligands against EsxA. Clustering analysis on the docking results led to the prediction of 5 important binding sites on EsxA. Out of the top 100 docking score compounds, 5 hit compounds were validated by secondary screening using NMR titration experiment. One hit compound (6058448) showed a MIC of 25uM by broth microdilution assay and one hit compound (5674203) showed anti virulence effects by inhibiting the expression of protein A and alpha-toxin virulence factors of SA. The present study using the in-silico structure-based screening platform has laid the foundation for drug development targeting EsxA. The resulting confirmed hit compounds will be useful leads to develop therapeutic drugs to combat MRSA.
Keywords: EsxA; Virulence Factor; Staphylococcus Aureus; MRSA; In Silico Structure-Based Screening; NMR Titration and global threat, in particular the number of both hospital-acquired and community-acquired cases are rising, leading to many life-threatening diseases such as endocarditis, pneumonia and toxic shock syndrome [3]. MRSA has been endemic in Hong Kong since the mid-1980s. Approximately 70% and 58% of the total and blood culture isolates of S. aureus in Hong Kong public hospitals are MRSA, respectively [4]. MRSA is getting more resistant to antibiotics that are currently used, and even a glycopeptides resistant strain, Vancomycin-resistant S. aureus (VRSA) has also been reported [5]. Similarly, MTB, the causative agent of tuberculosis, is still a major potential threat. Globally, an estimation of 9.6 million people had suffered from TB, and among these, about 16%, or 1.5 million, were killed [6]. It was estimated that 3.3% of new TB cases are multidrug-resistant tuberculosis (MDR-TB), and 20% of previously treated cases are MDR-TB, which are significant numbers [6]. MDR-TB is extremely difficult to cure, with a treatment success rate of merely 50% [6]. Furthermore, an even more resistant form of TB, totally drug-resistant TB (TDR-TB) has been described [7].
With resistance to all tested drugs, TDR-TB might be untreatable by any currently available drugs [8]. Therefore, there is a definite need to develop new approaches and druggable targets for combating drug-resistant pathogens.
A novel approach to solve the problem of drug-resistance is to "disarm" the microbes [9]. Instead of being bactericidal or bacteriostatic, the new drugs would inhibit virulence factors of pathogens, and control the infection only by lowering the virulence of the pathogens. Virulence of a pathogen refers to its ability to cause diseases and is contributed by a variety of factors that lead to damage in the host [10]. These factors, for example, include molecules that assist in host-recognition, toxins that damage host tissue, adhesins that assist adhesion, and secretion system-related molecules that assist the release of other factors [10,11]. S. aureus pathogenesis depends on a specialized protein secretion system (type VII-like Ess) that delivers a range of virulence factors to assist infectivity by establishment of abscess lesions and suppression of host immune responses [12,13]. EsxA is a confirmed excretory virulence factor of S. aureus [14]. EsxA is reported to be an immune evasion gene but how it can achieve this function is still unclear [15]. EsxA is small alpha-helical polypeptides belonging to a family of WXG100 motif proteins [16], which have a size of approximately 100 amino acids containing a helical structure and a conserved Trp-Xaa-Gly (WXG) motif [17]. ESAT-6 (homolog of S. aureus EsxA) and CFP-10 (homolog of S. aureus EsxB), secreted by Mycobacterium tuberculosis (MTB), are the founding members of the WXG100 motif family. ESAT-6 and CFP-10 are early secreted virulence factors by Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) [18,19]. These virulence factors are crucial for the replication of MTB in macrophages and presumably also for the pathogen's ability to suppress innate and adaptive immune responses [19][20][21]. Mutants that fail to secrete EsxA or EsxB display defects in the pathogenesis of S. aureus murine abscesses, suggesting that these specialized secreted WXG100 motif proteins may be a general strategy of human bacterial pathogenesis [14]. Therefore, EsxA is a promising drug target for combating MRSA and drugs targeting EsxA may have "add-on" implications in fighting against other drug resistant pathogens such as multidrug-resistant strains of MTB. In the present study we have streamlined a platform to perform in silico structure-based screening on a large ligand library containing 6.8 million compounds to identify lead compounds targeting the virulence factor EsxA of S. aureus.

Materials and Methods
In Silico Structure-Based Screening on EsxA and

Clustering Analysis
In silico structure-based virtual screening on S. aureus EsxA

NMR Titration Experiments of EsxA with Hit Compounds
To experimentally validate the docking results, the protein-

Virulence Assay on Validated List of EsxA Hit Compounds
Given that EsxA is a virulence factor of S. aureus, the effect of the validated hit compounds on the virulence of MRSA was tested by observing the change in expression of virulence genes. Two types of transformed bacteria were prepared, US300-pGLspa2 and US300-pGLhla. They are the MRSA strains US300, transformed by luciferase plasmids encoding Staphylococcal protein A promoter (pGLspa2) and alpha-toxin promoter (pGLhla), respectively. As the plasmids also encode a chloramphenicol resistance gene, the bacteria culture stock from -80 °C freezer were streaked onto BHI agar containing 10 ug/mL chloramphenicol incubated at 37 °C for 16h for selection. 5 isolated colonies from each type of bacteria were then picked and cultured in 2mL BHI with 10ug/mL chloramphenicol for 16-18h in 37 °C shaker. The lux was then measured for each type of the cultured bacteria, and the tube giving highest lux was taken to culture for a further 6h at 37 °C shaker. Then, disk diffusion test sterile paper disks were prepared and aligned on plates spread with the test bacteria. Lastly, after incubating the plates at 37 °C for 16h, the results were visualized using PerkinElmer IVIS Spectrum In Vivo Imaging System.

Results and Discussion
In Silico Structure-Based Screening on EsxA and

Clustering Analysis
In the present study, a dedicated research platform with the capacity for performing in silico structure-based screening of large ligand library on a target virulence factor protein was setup.
Autodock Vina dockings were completed on all 6.8 million ligands in the library against the X-ray structure of EsxA (PDB 2VS0). Docking score sorting and clustering analysis by AuPosSOM of the best-hit ligands according to their structural features and poses were performed in order to draw up the list of best docking scoring ligands for secondary screening and validation. Figure   1A-1D showed the AuPosSOM scoring plots and the graphical representation of the major predicted contacts on EsxA dimer structure.

NMR Titration Experiments of EsxA with Hit Compounds
Out of the 100 top hit compounds, 5 compounds with IDs and IUPAC names shown in Figure 1E   AuPosSOM scoring plot using all contacts for analysis. Each leaf number corresponds to a particular cluster of compounds with similar binding modes. A high positive score indicate high chance of finding active compounds inside the leaf; B) AuPosSOM scoring plot using hydrogen bonding contacts for analysis. C) Graphical representation of major predicted contacts on surface plot of EsxA dimer structure (PDB: 2VS0). D) Graphical representation of major predicted contacts in ribbon plot. Red: Major contacts exclusive to leaf 6 of all contacts main tree; Green: Major contacts exclusive to leaf 7 of all contacts main tree; Yellow: Major contacts discovered in both highest scoring leaf 6 and 7 of all contacts main tree; Cyan: Major contacts that are exclusively found in hydrogen bond analysis.

E)
List of hit compounds that displayed binding effect in secondary experiment screen by NMR titration assay with EsxA.

Susceptibility Tests on List of EsxA Hit Compounds
The broth microdilution test showed that hit compound 6058448 showed MIC at 25 uM while hit compounds 5224045, 5546503, and 6238413 have MIC >100 uM (Supplementary Figure   S2).  Top panel is the inhibition assays against US300-pGLspa2 transformed by luciferase plasmids encoding protein A promoter at 5mM and 500uM. Bottom panel is the inhibition assays against US300-pGLhla transformed by luciferase plasmids encoding alpha-toxin promoter at 5mM and 500uM.

Conclusion
The local and global spread of MRSA is of great concern because it is quickly acquiring resistance to all clinical antibacterial agents. 13854A factor of S. aureus. For example, hit compound 5674203 showed no antibacterial effect but significant anti-virulence effects to enhance host cell protection. Anti-virulence drugs against S. aureus infection with diminished virulence will cause less or even no damage to the host cells and tissues and will not subject to natural selection pressure so that the anti-virulence drug will less likely cause drug resistance. Through the hit-to-lead process, these hit compounds can be further modified and improved to attain higher potency and better pharmacokinetics properties. The improved compounds could be the candidates for new drug development against MRSA.
Through the same approach, we have also obtained promising hit compounds against the ESAT-6 and CFP-10 virulence factors of MTB, which can effectively suppress the intracellular survival of MTB model bacteria Mycobacterium marinum in macrophages without directly killing the bacteria (to be published in a forthcoming manuscript). Therefore, the present platform for in silico structurebased screening of large ligand library can be readily applied to other important drug targets or virulence factors with known highresolution structures.