Promising Antibacterial Target Research Articles

Development of Penicillin-Based Carbonic Anhydrase Inhibitors Targeting Multidrug-Resistant Neisseria gonorrhoeae.

The development of antibacterial drugs with new mechanisms of action is crucial in combating the rise of antibiotic-resistant infections. Bacterial carbonic anhydrases (CAs, EC 4.2.1.1) have been validated as promising antibacterial targets against pathogens such as Helicobacter pylori, Neisseria gonorrhoeae, and vancomycin-resistant enterococci. A multitarget strategy is proposed to design penicillin-based CA inhibitor hybrids for tackling resistance by targeting multiple bacterial pathways, thereby resensitizing drug-resistant strains to clinical antibiotics. The sulfonamide derivatives potently inhibited the CAs from N. gonorrhoeae and Escherichia coli with KI values in the range of 7.1-617.2 nM. Computational simulations with the main penicillin-binding protein (PBP) of N. gonorrhoeae indicated that these hybrid derivatives maintained the mechanism of action of the lead β-lactams. A subset of derivatives showed potent PBP-related antigonococcal effects against multidrug-resistant N. gonorrhoeae strains, with several compounds significantly outperforming both the lead β-lactam and CA inhibitor drugs (MIC values in the range 0.25 to 0.5 μg/mL).

An in silico approach for identification of lead compound as FtsZ inhibitor.

The remarkable conservation of the FtsZ among Gram-positive and Gram-negative bacteria, a crucial GTPase in bacterial cell division, has emerged as a promising antibacterial drug target to combat antibacterial resistance. There have been several coordinated efforts to develop inhibitors against FtsZ which can also serve as potential candidates for future antibiotics. In the present study, a natural product-like library (≈50,000 compounds) was employed to conduct HTVS against Staphylococcus aureus FtsZ protein (PDB Id: 6KVP). Additionally, molecular docking was carried out in two modes, SP and XP docking, using the Schrödinger suite. The glide scores of ligands obtained by XP docking were further summarized and compared with the control ligands (ZI1- co-crystal and PC190723-a compound undergoing clinical trial). Using the Prime-MM-GBSA approach, BFE calculations were performed on the top XP-scored ligands (≈598 compounds). These hits were also evaluated for ADMET parameters using the Qikprop algorithm, SwissADME, and in silico carcinogenicity testing using Carcinopred-El. Based on the results, ligand 4-FtsZ complex was considered for the 300ns MDS analysis to get insights into its binding modes within the catalytic pocket of FtsZ protein. The analysis revealed that the amide linkage sandwiched between the triazole and 1-oxa-8-azaspirodecan-8-ium moiety (Val203) as well as the aminoethyl group present at 1st position on the triazole moiety (Leu209, Leu200, Asp210, and Ala202) were responsible for the FtsZ inhibitory activity, owing to their crucial interactions with key amino acid residues. Further, the complex also displayed good protein-ligand stability, ultimately predicting ligand 4 as a potent lead compound for the inhibition of FtsZ. Thus, our in silico findings will serve as a framework for in-depth in-vitro and in-vivo investigations encouraging the development of FtsZ inhibitors as a new generation of antibacterial agents.

T5S1607 identified as a antibacterial FtsZ inhibitor：Virtual screening combined with bioactivity evaluation for the drug discovery

Due to antibiotic overuse, many bacteria have developed resistance, creating an urgent need for novel antimicrobial agents. It has been established that the filamentous temperature-sensitive mutant Z (FtsZ) of the bacterial cell division protein is an effective and promising antibacterial target. In this study, the optimal proteins were assessed by early recognition ability and the processed compound libraries were virtually screened using Vina. This effort resulted in the identification of 14 potentially active antimicrobial compounds. Among them, the compound T5S1607 demonstrated remarkable antibacterial efficacy against Bacillus subtilis ATCC9732 (MIC = 1 μg/mL) and Staphylococcus aureus ATC5C6538 (MIC = 4 μg/mL). Furthermore, in vitro experiments demonstrated that the selected compound T5S1607 rapidly killed bacteria and induced FtsZ protein aggregation, preventing bacterial division and leading to bacterial death. Additionally, cell toxicity and hemolysis experiments indicate that compound T5S1607 exhibits minimal toxicity to LO2 cells and shows no significant hemolytic effects on mammalian cells in vitro at the MIC concentration range. All the results indicate that compound T5S1607 is a promising antibacterial agent and a potential FtsZ inhibitor. In conclusion, this work successfully discovered FtsZ inhibitors with good activity through the virtual screening drug discovery process.

Structure of Staphylococcus aureus ClpP Bound to the Covalent Active‐Site Inhibitor Cystargolide A

AbstractThe caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural β‐lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti‐virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by β‐lactones, the predominant class of ClpP inhibitors.

Structure of Staphylococcus aureus ClpP Bound to the Covalent Active-Site Inhibitor Cystargolide A.

The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural β-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by β-lactones, the predominant class of ClpP inhibitors.

Interactome Analysis Identifies MSMEI_3879 as a Substrate of Mycolicibacterium smegmatis ClpC1.

The prevalence of drug-resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new drug targets in this globally important pathogen. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize compounds that impinge on ClpC1 activity are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a coimmunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a surrogate for M. tuberculosis. We identify a diverse panel of interaction partners, many of which coimmunoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis establishes MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires exposure of its N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs on substrates. Fluorescent substrates incorporating MSMEI_3879 may be useful in screening for novel ClpC1-targeting antibiotics to help address the challenge of M. tuberculosis drug resistance. IMPORTANCE Drug-resistant tuberculosis infections are a major challenge to global public health. Much effort has been invested in identifying new drug targets in the causative pathogen, Mycobacterium tuberculosis. One such target is the ClpC1 unfoldase. Compounds have been identified that kill M. tuberculosis by disrupting ClpC1 activity, yet the physiological function of ClpC1 in cells has remained poorly defined. Here, we identify interaction partners of ClpC1 in a model mycobacterium. By building a broader understanding of the role of this prospective drug target, we can more effectively develop compounds that inhibit its essential cellular activities.

Anti-vibriosis bioactive molecules from Arctic Penicillium sp. Z2230

Vibrio species (Vibrio sp.) is a class of Gram-negative aquatic bacteria that causes vibriosis in aquaculture, which have resulted in big economic losses. Utilization of antibiotics against vibriosis has brought concerns on antibiotic resistance, and it is essential to explore potential antibiotic alternatives. In this study, seven compounds (compounds 1–7) were isolated from the Arctic endophytic fungus Penicillium sp. Z2230, among which compounds 3, 4, and 5 showed anti-Vibrio activity. The structures of the seven compounds were comprehensively elucidated, and the antibacterial mechanism of compounds 3, 4, and 5 was explored by molecular docking. The results suggested that the anti-Vibrio activity could come from inhibition of the bacterial peptide deformylase (PDF). This study discovered three Penicillium-derived compounds to be potential lead molecules for developing novel anti-Vibrio agents, and identified PDF as a promising antibacterial target. It also expanded the bioactive diversity of polar endophytic fungi by showing an example in which the secondary metabolites of a polar microbe were a good source of natural medicine.Graphical

Synthesis, molecular docking and antibacterial activity of an oxadiazole-based lipoteichoic acid inhibitor and its metabolites

Amongst drug resistant Gram-positive bacteria, Staphylococcus aureus is a pathogen of great concern as it is the leading cause of life-threatening nosocomial and community acquired infections which are often associated with implanted medical devices. The biosynthesis of lipotheicoic acid (LTA) by S. aureus has been recognized as a promising antibacterial target, owing its critical role in the growth and survival of Gram-positive bacteria. Here we report for the first time the chemical synthesis and characterisation of an oxadiazole based compound (1771), previously described as an inhibitor of LTA biosynthesis by targeting Lta synthase enzyme (LtaS). To investigate its controversial mode of action, we also performed molecular docking studies, which indicated that 1771 behaves as a competitive inhibitor against LtaS. We also synthesised and evaluated the antimicrobial activity of 1771 metabolites which we have identified from its decomposition in mouse serum, proving that the biological activity was caused by intact 1771.

Computer-aided design of a cyclic di-AMP synthesizing enzyme CdaA inhibitor.

Cyclic di-AMP (c-di-AMP) is an essential secondary messenger regulating cell wall homeostasis and myriads of physiological processes in several Gram-positive and mycobacteria, including human pathogens. Hence, c-di-AMP synthesizing enzymes (DACs) have become a promising antibacterial drug target. To overcome a scarcity of small molecule inhibitors of c-di-AMP synthesizing enzyme CdaA, a computer-aided design of a new compound that should block the enzyme has been performed. This has led to the identification of a molecule comprising two thiazole rings and showing inhibitory potential based on ITC measurements. Thiazole scaffold is a good pharmacophore nucleus known due to its various pharmaceutical applications. It is contained in more than 18 FDA-approved drugs as well as in dozens of experimental drugs. Hence, the designed inhibitor can serve as a potent lead compound for further development of inhibitor against CdaA.

Structure-Based Ligand Design Targeting Pseudomonas aeruginosa LpxA in Lipid A Biosynthesis.

Enzymes involved in lipid A biosynthesis are promising antibacterial drug targets in Gram-negative bacteria. In this study, we use a structure-based design approach to develop a series of novel tetrazole ligands with low μM affinity for LpxA, the first enzyme in the lipid A pathway. Aided by previous structural data, X-ray crystallography, and surface plasmon resonance bioanalysis, we identify 17 hit compounds. Two of these hits were subsequently modified to optimize interactions with three regions of the LpxA active site. This strategy ultimately led to the discovery of ligand L13, which had a KD of 3.0 μM. The results reveal new chemical scaffolds as potential LpxA inhibitors, important binding features for ligand optimization, and protein conformational changes in response to ligand binding. Specifically, they show that a tetrazole ring is well-accommodated in a small cleft formed between Met169, the "hydrophobic-ruler" and His156, both of which demonstrate significant conformational flexibility. Furthermore, we find that the acyl-chain binding pocket is the most tractable region of the active site for realizing affinity gains and, along with a neighboring patch of hydrophobic residues, preferentially binds aliphatic and aromatic groups. The results presented herein provide valuable chemical and structural information for future inhibitor discovery against this important antibacterial drug target.

TXH11106: A Third-Generation MreB Inhibitor with Enhanced Activity against a Broad Range of Gram-Negative Bacterial Pathogens.

The emergence of multi-drug-resistant Gram-negative pathogens highlights an urgent clinical need to explore and develop new antibiotics with novel antibacterial targets. MreB is a promising antibacterial target that functions as an essential elongasome protein in most Gram-negative bacterial rods. Here, we describe a third-generation MreB inhibitor (TXH11106) with enhanced bactericidal activity versus the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa compared to the first- and second-generation compounds A22 and CBR-4830, respectively. Large inocula of these four pathogens are associated with a low frequency of resistance (FOR) to TXH11106. The enhanced bactericidal activity of TXH11106 relative to A22 and CBR-4830 correlates with a correspondingly enhanced capacity to inhibit E. coli MreB ATPase activity via a noncompetitive mechanism. Morphological changes induced by TXH11106 in E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa provide further evidence supporting MreB as the bactericidal target of the compound. Taken together, our results highlight the potential of TXH11106 as an MreB inhibitor with activity against a broad spectrum of Gram-negative bacterial pathogens of acute clinical importance.

Structure and role of the linker domain of the iron surface-determinant protein IsdH in heme transportation in Staphylococcus aureus

Staphylococcus aureus is a major cause of deadly nosocomial infections, a severe problem fueled by the steady increase of resistant bacteria. The iron surface determinant (Isd) system is a family of proteins that acquire nutritional iron from the host organism, helping the bacterium to proliferate during infection, and therefore represents a promising antibacterial target. In particular, the surface protein IsdH captures hemoglobin (Hb) and acquires the heme moiety containing the iron atom. Structurally, IsdH comprises three distinctive NEAr-iron Transporter (NEAT) domains connected by linker domains. The objective of this study was to characterize the linker region between NEAT2 and NEAT3 from various biophysical viewpoints and thereby advance our understanding of its role in the molecular mechanism of heme extraction. We demonstrate the linker region contributes to the stability of the bound protein, likely influencing the flexibility and orientation of the NEAT3 domain in its interaction with Hb, but only exerts a modest contribution to the affinity of IsdH for heme. Based on these data, we suggest that the flexible nature of the linker facilitates the precise positioning of NEAT3 to acquire heme. In addition, we also found that residues His45 and His89 of Hb located in the heme transfer route toward IsdH do not play a critical role in the transfer rate-determining step. In conclusion, this study clarifies key elements of the mechanism of heme extraction of human Hb by IsdH, providing key insights into the Isd system and other protein systems containing NEAT domains.

Structural and functional analysis of the D-alanyl carrier protein ligase DltA from Staphylococcus aureus Mu50.

D-Alanylation of the teichoic acids of the Gram-positive bacterial cell wall plays crucial roles in bacterial physiology and virulence. Deprivation of D-alanine from the teichoic acids of Staphylococcus aureus impairs biofilm and colony formation, induces autolysis and ultimately renders methicillin-resistant S.aureus highly susceptible to antimicrobial agents and host defense peptides. Hence, the D-alanylation pathway has emerged as a promising antibacterial target against drug-resistant S. aureus. D-Alanylation of teichoic acids is mediated via the action of four proteins encoded by the dlt operon, DltABCD, all four of which are essential for the process. In order to develop novel antimicrobial agents against S. aureus, the D-alanyl carrier protein ligase DltA, which is the first protein in the D-alanylation pathway, was focused on. Here, the crystal structure of DltA from the methicillin-resistant S. aureus strain Mu50 is presented, which reveals the unique molecular details of the catalytic center and the role of the P-loop. Kinetic analysis shows that the enantioselectivity of S.aureus DltA is much higher than that of DltA from other species. In the presence of DltC, the enzymatic activity of DltA is increased by an order of magnitude, suggesting a new exploitable binding pocket. This discovery may pave the way for a new generation of treatments for drug-resistant S. aureus.

Structural basis for the allosteric inhibition of UMP kinase from Gram-positive bacteria, a promising antibacterial target.

Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate (UMP) kinase is a promising target for novel antibiotic discovery as it is essential for bacterial survival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regulation with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

High Affinity Iron Acquisition Systems Facilitate but Are Not Essential for Colonization of Chickens by Salmonella Enteritidis.

The roles of TonB mediated Fe3+ (ferric iron) uptake via enterobactin (involving biosynthesis genes entABCDEF) and Fe2+ (ferrous iron) uptake through the FeoABC transporter are poorly defined in the context of chicken-Salmonella interactions. Both uptake systems are believed to be the major contributors of iron supply in the Salmonella life cycle. Current evidence suggests that these iron uptake systems play a major role in pathogenesis in mammals and as such, they represent promising antibacterial targets with therapeutic potential. We investigated the role of these iron uptake mechanisms regarding the ability of Salmonella Enteritidis (SEn) strains to colonize in a chicken infection model. Further we constructed a bioluminescent reporter to sense iron limitation during gastrointestinal colonization of Salmonella in chicken via ex vivo imaging. Our data indicated that there is some redundancy between the ferric and ferrous iron uptake mechanisms regarding iron acquisition during SEn pathogenesis in chicken. We believe that this redundancy of iron acquisition in the host reservoir may be the consequence of adaptation to unique avian environments, and thus warrants further investigation. To our knowledge, this the first report providing direct evidence that both enterobactin synthesis and FeoABC mediated iron uptake contribute to the virulence of SEn in chickens.

Improving Antibacterial Activity of a HtrA Protease Inhibitor JO146 against Helicobacter pylori: A Novel Approach Using Microfluidics-Engineered PLGA Nanoparticles.

Nanoparticle drug delivery systems have emerged as a promising strategy for overcoming limitations of antimicrobial drugs such as stability, bioavailability, and insufficient exposure to the hard-to-reach bacterial drug targets. Although size is a vital colloidal feature of nanoparticles that governs biological interactions, the absence of well-defined size control technology has hampered the investigation of optimal nanoparticle size for targeting bacterial cells. Previously, we identified a lead antichlamydial compound JO146 against the high temperature requirement A (HtrA) protease, a promising antibacterial target involved in protein quality control and virulence. Here, we reveal that JO146 was active against Helicobacter pylori with a minimum bactericidal concentration of 18.8–75.2 µg/mL. Microfluidic technology using a design of experiments approach was utilized to formulate JO146-loaded poly(lactic-co-glycolic) acid nanoparticles and explore the effect of the nanoparticle size on drug delivery. JO146-loaded nanoparticles of three different sizes (90, 150, and 220 nm) were formulated with uniform particle size distribution and drug encapsulation efficiency of up to 25%. In in vitro microdilution inhibition assays, 90 nm nanoparticles improved the minimum bactericidal concentration of JO146 two-fold against H. pylori compared to the free drug alone, highlighting that controlled engineering of nanoparticle size is important in drug delivery optimization.

The discovery of natural 4'-demethylepipodophyllotoxin from renewable Dysosma versipellis species as a novel bacterial cell division inhibitor for controlling intractable diseases in rice

Natural products (NPs) are inexhaustible wellsprings for new agrochemicals discovery. To excavate novel natural products-based antibacterial leads or candidates potentially targeting FtsZ (filamentous temperature-sensitive protein Z) which is widely regarded as a promising antibacterial target for novel drug discovery, herein, a rapid and efficient screening platform was initially established to disclose the potential FtsZ inhibitors from a variety of commercial natural products. Screening results revealed that 4'-demethylepipodophyllotoxin (DMEP) from renewable Dysosma versipellis (D. versipellis) species could distinctly decrease the XooFtsZ’ GTPase activity and afforded a ponderable bacteriostatic effect (EC50 =38.7 mg L-1) against Xanthomonas oryzae pv. oryzae (Xoo). Further explorations found that DMEP could disorganize the assembly dynamics of XooFtsZ, and thereby yielding long and filamentous Xoo cells from the transmission electron microscopic and fluorescence microscopic imaging. Molecular docking study showed that DMEP displayed strong interactions with Asp38 and Arg205 residues of XooFtsZ, indicating that a natural potential FtsZ inhibitor was discovered gradually. The following bioassay result against rice bacterial blight in vivo presented good curative activity (50.0% at 200 mg L−1) and protective activity (46.8% at 200 mg L−1). Given that natural DMEP has many sites for structural modification, therefore, it can concurrently serve as a novel lead compound for exploring highly bioactive FtsZ inhibitors based on the molecular skeleton of DMEP.

Opportunities and Challenges of Bacterial Glycosylation for the Development of Novel Antibacterial Strategies.

Glycosylation is a ubiquitous process that is universally conserved in nature. The various products of glycosylation, such as polysaccharides, glycoproteins, and glycolipids, perform a myriad of intra- and extracellular functions. The multitude of roles performed by these molecules is reflected in the significant diversity of glycan structures and linkages found in eukaryotes and prokaryotes. Importantly, glycosylation is highly relevant for the virulence of many bacterial pathogens. Various surface-associated glycoconjugates have been identified in bacteria that promote infectious behavior and survival in the host through motility, adhesion, molecular mimicry, and immune system manipulation. Interestingly, bacterial glycosylation systems that produce these virulence factors frequently feature rare monosaccharides and unusual glycosylation mechanisms. Owing to their marked difference from human glycosylation, bacterial glycosylation systems constitute promising antibacterial targets. With the rise of antibiotic resistance and depletion of the antibiotic pipeline, novel drug targets are urgently needed. Bacteria-specific glycosylation systems are especially promising for antivirulence therapies that do not eliminate a bacterial population, but rather alleviate its pathogenesis. In this review, we describe a selection of unique glycosylation systems in bacterial pathogens and their role in bacterial homeostasis and infection, with a focus on virulence factors. In addition, recent advances to inhibit the enzymes involved in these glycosylation systems and target the bacterial glycan structures directly will be highlighted. Together, this review provides an overview of the current status and promise for the future of using bacterial glycosylation to develop novel antibacterial strategies.

In-silico driven design and development of spirobenzimidazo-quinazolines as potential DNA gyrase inhibitors

DNA gyrase and Topoisomerase IV are promising antibacterial drug targets as they regulate bacterial DNA replication and topology. In a quest for novel DNA topoisomerase inhibitors, a multidisciplinary approach was adopted that involves computational prediction of binding sites and molecular modelling followed by green synthesis and biological evaluation of antibacterial activity of spirobenzimidazo quinazolines derivatives. Using basic quantum chemistry principles, we evaluated spirobenzimidazo quinazolines derivatives with their pharmacokinetic profiles. Based on the results of the aforesaid in-silico studies, we synthesized a series of titled compounds using green synthetic methodology that were validated as potential antimicrobial agents. Quantum chemoinformatics based predicted activity for the synthesized compounds 9b, 9c, and 9j was concomitant with biological evaluation of broadspectrum antibacterial activity. Biological evaluation revealed that inhibition of biofilm formation was due to their potential antibacterial activity. We believe that the novel spirobenzimidazo quinazolines have the potential to be alternatives to aminocoumarins and classical quinazolines upon detailed target specific biological studies.

In silico evaluation of antimicrobial, antihyaluronidase and bioavailability parameters of rosmarinic acid in Perilla frutescens leaf extracts

The present study reports ultrasound-assisted extraction of rosmarinic acid extract using the leaves of Perilla frutescens L. by using ethanol solvent. P. frutescens rosmarinic acid extract (PF-RAE) exhibited antimicrobial activity against bacteria and fungi. PF-RAE exhibited excellent antioxidant activity against DPPH and ABTS radicals. PF-RAE exhibited significant in vitro hyaluronidase inhibitory activity with maximum inhibition of 44.35%. HPLC analysis showed that rosmarinic acid is the major component of PF-RAE and it is responsible for antimicrobial, antioxidant and hyaluronidase inhibitory activities. Further, we report some of the plausible mechanisms of multiple biological functions of rosmarinic acid by in silico studies. Molecular docking studies reported that rosmarinic acid strongly inhibits bacterial co-/posttranslation enzyme peptide deformylase (promising antibacterial target) as well as fungi posttranslation enzyme N-myristoyltransferase (promising antifungal target) with binding affinities of 7.9 and 7.1 Kcal/mol, respectively. Further, rosmarinic acid exhibited strong affinity with human hyaluronidase protein by H-bonding interactions. Rosmarinic acid satisfactorily follows the Lipinski’s rule of five with ‘0’ violations, showed excellent hydrophilicity, oral bioavailability, skin permeability and gastrointestinal permeability and showed no toxicity at all. We concluded that rosmarinic acid can be developed as an excellent pharmaceutical and nutraceutical drug ingredient.