Low-Generation Cationic Phosphorus Dendrimers: Novel Approach to Tackle Drug-Resistant S. aureus In Vitro and In Vivo.

The incessant, global increase in antimicrobial resistance (AMR) is a very big challenge for healthcare systems. AMR is predicted to grow at an alarming pace, with a dramatic increase in morbidity, mortality, and a 100 trillion US$ loss to the global economy by 2050. The mortality rate caused by methicillin-resistant S. aureus (MRSA) is much higher as compared to infections caused by drug-susceptible S. aureus. Additionally, there is a big paucity of therapeutics available for treatment of serious infections caused by MRSA. Thus, the discovery and development of novel therapies is an urgent, unmet medical need. In this context, we synthesized AE4G0, a low-generation cationic-phosphorus dendrimer expressing potent antimicrobial activity against S. aureus and Enterococcus sp., and demonstrating a broad selectivity index against eukaryotic cells. AE4G0 exhibits concentration-dependent, bactericidal activity and synergizes with gentamicin, especially against gentamicin-resistant MRSA NRS119. Fluorescence and scanning electron microscopy demonstrate that treatment with AE4G0 led to the utter destruction of S. aureus ATCC 29213 without inducing resistance, despite repeated exposure. When tested in vivo, AE4G0 demonstrates significant efficacy against S. aureus ATCC 29213, alone and in combination with gentamicin against gentamicin-resistant S. aureus NRS119 in the murine skin model of infection. Taken together, AE4G0 demonstrates the potential to be translated as a novel therapeutic option for the treatment of topical, drug-resistant S. aureus infections.

Exploration of Potent Antiviral Phytomedicines from Lauraceae Family Plants against SARS-CoV-2 Main Protease.

A new Coronaviridae strain, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), emerged from Wuhan city of China and caused one of the substantial global health calamities in December 2019. Even though several vaccines and drugs have been developed worldwide since COVID-19, a cost-effective drug with the least side effects is still unavailable. Currently, plant-derived compounds are mostly preferred to develop antiviral therapeutics due to its less toxicity, easy access, and cost-effective characteristics. Therefore, in this study, 124 phytochemical compounds from plants of Lauraceae family with medicinal properties were virtually screened against SARS-CoV-2 Mpro. Identification of four phytomolecules, i.e., cassameridine, laetanine, litseferine and cassythicine, with docking scores -9.3, -8.8, -8.6, and -8.6 kcal/mol, respectively, were undertaken by virtual screening, and molecular docking. Furthermore, the molecular dynamic simulation and essential dynamics analysis have contributed in understanding the stability and inhibitory effect of these selected compounds. These phytomolecules can be considered for further in vitro and in vivo experimental study to develop anti-SARS-CoV-2 therapeutics targeting the main protease (Mpro).

Developing the Natural Prenylflavone Artocarpin from Artocarpus hirsutus as a Potential Lead Targeting Pathogenic, Multidrug-Resistant Staphylococcus aureus, Persisters and Biofilms with No Detectable Resistance.

The genus Artocarpus, a nutraceutical, is widely used in traditional medicine for treatment of many chronic diseases including infections. Artocarpus hirsutus Lam., an evergreen tree endogenous to the Western Ghats of India, is a well-documented medicinal plant in Hortus Malabaricus, the oldest comprehensive printed book on the natural plant wealth of Asia. Herein we describe artocarpin, a major isoprenyl flavonoid isolated from the stem bark of A. hirsutus Lam., as the explanation behind the indigenous knowledge reported for treatment of various skin ailments. Artocarpin, a noncytotoxic, isoprenyl flavonoid, is rapidly bactericidal against multiple World Health Organization (WHO) priority 2 pathogens including multidrug-resistant Staphylococcus aureus and Enterococcus sp. with an extended postantibiotic effect. Artocarpin (AH-5) synergizes with gentamicin and linezolid, inhibits bacteria in different physiological states, including under biofilm and in macrophages, and does not induce resistance in S. aureus despite repeated exposure. Artocarpin induces rapid cellular lysis, as confirmed by fluorescence microscopy and scanning electron microscopy analysis as well as by measuring the significantly increased extracellular and concomitantly decreased intracellular adenosine triphosphate levels. When tested in vivo, AH-5 is almost as effective as vancomycin in reducing bacterial load in murine thigh and skin infection models, which is comparable to standard of care (SoC) antibiotics. This is highly significant since AH-5 is a direct natural entity that has been evaluated without any pharmaceutical modification and expresses robust in vitro and in vivo antibacterial activity, which is comparable to highly optimized SoC comparators and further could be considered as an effective clinical, antibacterial drug lead.

Potential Efficacy of ß-Amyrin Targeting Mycobacterial Universal Stress Protein by In Vitro and In Silico Approach.

The emergence of drug resistance and the limited number of approved antitubercular drugs prompted identification and development of new antitubercular compounds to cure Tuberculosis (TB). In this work, an attempt was made to identify potential natural compounds that target mycobacterial proteins. Three plant extracts (A. aspera, C. gigantea and C. procera) were investigated. The ethyl acetate fraction of the aerial part of A. aspera and the flower ash of C. gigantea were found to be effective against M. tuberculosis H37Rv. Furthermore, the GC-MS analysis of the plant fractions confirmed the presence of active compounds in the extracts. The Mycobacterium target proteins, i.e., available PDB dataset proteins and proteins classified in virulence, detoxification, and adaptation, were investigated. A total of ten target proteins were shortlisted for further study, identified as follows: BpoC, RipA, MazF4, RipD, TB15.3, VapC15, VapC20, VapC21, TB31.7, and MazF9. Molecular docking studies showed that ß-amyrin interacted with most of these proteins and its highest binding affinity was observed with Mycobacterium Rv1636 (TB15.3) protein. The stability of the protein-ligand complex was assessed by molecular dynamic simulation, which confirmed that ß-amyrin most firmly interacted with Rv1636 protein. Rv1636 is a universal stress protein, which regulates Mycobacterium growth in different stress conditions and, thus, targeting Rv1636 makes M. tuberculosis vulnerable to host-derived stress conditions.

Repurposing disulfiram for treatment of Staphylococcus aureus infections.

BACKGROUND: Antimicrobial resistance is an urgent threat affecting healthcare systems worldwide. Identification of novel molecules capable of escaping current resistance mechanisms and exhibiting potent activity against highly drug-resistant strains is the unmet need of the hour. METHODS: Whole cell growth inhibition assays were used to screen and identify novel inhibitors. The hit compounds were tested against Vero cells to determine the selectivity index, followed by time-kill kinetics against Staphylococcus aureus. The ability of disulfiram to synergize with several approved drugs utilized for the treatment of S. aureus was determined using fractional inhibitory concentration indexes, followed by its ability to decimate staphyloccocal infections ex vivo. Finally, the in-vivo potential of disulfiram was determined in a neutropenic murine model of S. aureus infection. RESULTS: The screening showed that disulfiram has equipotent antibacterial activity against S. aureus, including clinical drug-resistant strains (minimum inhibitory concentration 8-16 mg/L). Disulfiram exhibited concentration-dependent bactericidal activity (∼7 log10 colony-forming units/mL reduction), synergized with linezolid and gentamycin against S. aureus, eradicated staphylococcal biofilms (64-fold better than vancomycin), decimated intracellular S. aureus better than vancomycin, exhibited longer post antibiotic effect than vancomycin, and reduced bacterial counts in murine thigh as well as vancomycin at 50 mg/kg. CONCLUSION: Taken together, disulfiram exhibits all the characteristics required for repurposing as an antibacterial targeting staphylococcal infections.

New fluoroquinolone compounds with endo-nortropine derivatives at C-7 position show antibacterial activity against fluoroquinolone-resistant strains of Staphylococcus aureus.

A series of new fluoroquinolone analogs (3-18) were prepared, in three steps, by substituting chloro esters and esters with cyclic amines on the C-7 endo-nortropine derivatives of difluoroquinolone acid. All the synthesized compounds displayed good MIC against the Staphylococcus aureus when initially screened for Escherichia coli, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The molecules were further evaluated for their antibacterial activity against fluoroquinolone-resistant strains of S. aureus and for cytotoxic assay. Based on the results, five of the sixteen compounds displayed the potential to be developed further for treatment against fluoroquinolone-resistant strains of S. aureus.

Repurposing disulfiram to target infections caused by non-tuberculous mycobacteria.

BACKGROUND: Non-tuberculous mycobacteria are emerging pathogens of significant worldwide interest because they have inherent drug resistance to a wide variety of FDA-approved drugs and cause a broad range of serious infections. In order to identify new drugs active against non-tuberculous mycobacteria, we identified disulfiram, utilized for treatment of alcohol dependence, as exhibiting potent growth-inhibitory activity against non-tuberculous mycobacteria. METHODS: Whole-cell growth inhibition assays were used to screen and identify novel inhibitors. The hit compounds were tested against Vero cells to determine the selectivity index, and this was followed by determining time-kill kinetics against Mycobacterium fortuitum and Mycobacterium abscessus. Disulfiram's ability to synergize with several approved drugs utilized for the treatment of M. fortuitum and M. abscessus was determined using fractional inhibitory concentration indexes followed by determining its ability to reduce mycobacterial infections ex vivo. Finally, disulfiram's in vivo potential was determined in a neutropenic murine model mimicking mycobacterial infection. RESULTS: We identified disulfiram as possessing potent antimicrobial activity against non-tuberculous mycobacteria. Disulfiram exhibited concentration- and time-dependent bactericidal activity against M. fortuitum as well as against M. abscessus and synergized with all drugs utilized for their treatment. Additionally, disulfiram reduced bacterial load in macrophages in an intracellular killing assay better than amikacin. When tested in a murine neutropenic M. fortuitum infection model, disulfiram caused significant reduction in bacterial load in kidneys. CONCLUSIONS: Disulfiram exhibits all properties required for it to be repositioned as a novel anti-mycobacterial therapy and possesses a potentially new mechanism of action. Thus, it can be considered as a potent structural lead for the treatment of non-tuberculous mycobacterial infections.

Biocatalytic synthesis of diaryl disulphides and their bio-evaluation as potent inhibitors of drug-resistant Staphylococcus aureus.

Staphylococcus aureus is a WHO Priority II pathogen for its capability to cause acute to chronic infections and to resist antibiotics, thus severely impacting healthcare systems worldwide. In this context, it is urgently desired to discover novel molecules to thwart the continuing emergence of antimicrobial resistance. Disulphide containing small molecules has gained prominence as antibacterials. As their conventional synthesis requires tedious synthetic procedure and sometimes toxic reagents, a green and environmentally benign protocol for their synthesis has been developed through which a series of molecules were obtained and evaluated for antibacterial activity against ESKAPE pathogen panel. The hit compound was tested for cytotoxicity against Vero cells to determine its selectivity index and time-kill kinetics was determined. The activity of hit was determined against a panel of S. aureus multi-drug resistant clinical isolates. Also, its ability to synergize with FDA approved drugs was tested as was its ability to reduce biofilm. We identified bis(2-bromophenyl) disulphide (2t) as possessing equipotent antimicrobial activity against S. aureus including MRSA and VRSA strains. Further, 2t exhibited a selectivity index of 25 with concentration-dependent bactericidal activity, synergized with all drugs tested and significantly reduced preformed biofilm. Taken together, 2t exhibits all properties to be positioned as novel scaffold for anti-staphylococcal therapy.

Identification and bioevaluation of SRI-12742 as an antimicrobial agent against multidrug-resistant Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii (MDR-Ab) is one of the most significant nosocomial pathogens that is being increasingly isolated in healthcare settings worldwide. Owing to its inherent drug-resistant nature, coupled with its ability to readily acquire resistance to other antibiotic classes, there is a real dearth of antibiotics available to treat infections with MDR-Ab. A commercially available library was screened against MDR-Ab BAA-1605 to identify novel inhibitory molecules. The selectivity index of a hit was tested against Vero cells and in vitro efficacy was profiled against a panel of clinical MDR-Ab. The bacteriostatic or bactericidal nature was determined by time-kill experiments, and synergy with clinically approved drugs was determined by the chequerboard method. Additionally, in vivo efficacy was measured in a murine neutropenic A. baumannii thigh infection model. SRI-12742 was identified as a potent active hit, with a minimum inhibitory concentration (MIC) of 4 mg/L against BAA-1605. Its activity was then profiled against a MDR-Ab clinical strain panel (MICs 4 mg/L to >64 mg/L). SRI-12742 exhibited concentration-dependent bactericidal activity and caused an ca. 16 log10 CFU/mL reduction at 10 × MIC in 24 h, which is comparable with minocycline. In a murine neutropenic thigh infection model of A. baumannii infection, SRI-12742 reduced CFU counts by ca. 0.9 log10 CFU, which is comparable with polymyxin B. In addition, SRI-12742 synergised with all classes of antibiotics tested. SRI-12742 exhibits all of the criteria necessary to be positioned as a novel lead with potential to be deployed for the treatment of infections caused by MDR-Ab.

Biological evaluation of diphenyleneiodonium chloride (DPIC) as a potential drug candidate for treatment of non-tuberculous mycobacterial infections.

BACKGROUND: Novel drug discovery against non-tuberculous mycobacteria is beset with a large number of challenges including the existence of myriad innate drug resistance mechanisms as well as a lack of suitable animal models, which hinders effective translation. In order to identify molecules acting via novel mechanisms of action, we screened the Library of Pharmacologically Active Compounds against non-tuberculous mycobacteria to identify such compounds. METHODS: Whole-cell growth inhibition assays were used to screen and identify novel inhibitors. The hit compounds were tested for cytotoxicity against Vero cells to determine the selectivity index, and time-kill kinetics were determined against Mycobacterium fortuitum. The compound's ability to synergize with amikacin, ceftriaxone, ceftazidime and meropenem was determined using fractional inhibitory concentration indexes followed by its ability to decimate mycobacterial infections ex vivo. Finally, the in vivo potential was determined in a neutropenic murine model mimicking mycobacterial infection. RESULTS: We have identified diphenyleneiodonium chloride (DPIC), an NADPH/NADH oxidase inhibitor, as possessing potent antimicrobial activity against non-tuberculous mycobacteria. DPIC exhibited concentration-dependent bactericidal activity against M. fortuitum and synergized with amikacin, ceftriaxone, ceftazidime and meropenem. When tested in a murine neutropenic M. fortuitum infection model, DPIC caused a significant reduction in bacterial load in kidney and spleen. The reduction in bacterial count is comparable to amikacin at a 100-fold lower concentration. CONCLUSIONS: DPIC exhibits all properties to be repositioned as a novel anti-mycobacterial therapy and possesses a potentially new mechanism of action. Thus, it can be projected as a potential new therapeutic against ever-increasing non-tuberculous mycobacterial infections.

A systematic screen of FDA-approved drugs for inhibitors of biological threat agents.

BACKGROUND: The rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency. METHODOLOGY/PRINCIPAL FINDINGS: A large systematic effort to determine whether existing drugs can be used against high containment bacterial and viral pathogens is described. We assembled and screened 1012 FDA-approved drugs for off-label broad-spectrum efficacy against Bacillus anthracis; Francisella tularensis; Coxiella burnetii; and Ebola, Marburg, and Lassa fever viruses using in vitro cell culture assays. We found a variety of hits against two or more of these biological threat pathogens, which were validated in secondary assays. As expected, antibiotic compounds were highly active against bacterial agents, but we did not identify any non-antibiotic compounds with broad-spectrum antibacterial activity. Lomefloxacin and erythromycin were found to be the most potent compounds in vivo protecting mice against Bacillus anthracis challenge. While multiple virus-specific inhibitors were identified, the most noteworthy antiviral compound identified was chloroquine, which disrupted entry and replication of two or more viruses in vitro and protected mice against Ebola virus challenge in vivo. CONCLUSIONS/SIGNIFICANCE: The feasibility of repurposing existing drugs to face novel threats is demonstrated and this represents the first effort to apply this approach to high containment bacteria and viruses.

Mannich reaction derivatives of novobiocin with modulated physiochemical properties and their antibacterial activities.

Synthetic derivatives of the natural product antibiotic novobiocin were synthesized in order to improve their physiochemical properties. A Mannich reaction was used to introduce new side chains at a solvent-exposed position of the molecule, and a diverse panel of functional groups was evaluated at this position. Novobiocin and the new derivatives were tested for their binding to gyrase B and their antibacterial activities against Staphylococcus aureus, Mycobacterium tuberculosis, Francisella tularensis and Escherichia coli. While the new derivatives still bound the gyrase B protein potently (0.07-1.8 µM, IC(50)), they had significantly less antibacterial activity. Two compounds were identified with increased antibacterial activity against M. tuberculosis, with a minimum inhibitory concentration of 2.5 µg/ml.

Identification of antimicrobial activity among FDA-approved drugs for combating Mycobacterium abscessus and Mycobacterium chelonae.

OBJECTIVES: Rapidly growing mycobacteria have long been neglected in drug discovery efforts and this neglect is reflected in the paucity of therapeutic options available for diseases resulting from these infections. The purpose of this work is to identify new candidate drugs for treating non-tuberculous mycobacteria (NTM) by testing FDA-approved drugs for antimicrobial activity against Mycobacterium abscessus and Mycobacterium chelonae, two emerging NTM drug-resistant pathogens. METHODS: In this study, we screened 1040 FDA-approved drugs against M. abscessus and M. chelonae. RESULTS: Of the drugs screened, 32 compounds exhibited significant antimicrobial activity, with an MIC ≤ 8 mg/L, against M. chelonae, while only 7 compounds showed such activity against M. abscessus. Notably, neostigmine bromide and cinnarizine exhibited highly significant antimicrobial activity against M. chelonae, but had little potency against M. abscessus. Metronidazole and puromycin were the only drugs that acted equipotently against both strains, in decreasing order of effectiveness. CONCLUSIONS: The dearth of identified compounds active against M. abscessus exemplifies its ability to resist drugs as well as the resilience of rapidly growing NTM. Repurposing of approved drugs is a viable alternative to de novo drug discovery and development.

Expression, purification, and biochemical characterization of Mycobacterium tuberculosis aspartate decarboxylase, PanD.

Like all bacteria, Mycobacterium tuberculosis (Mtb) possesses the genes necessary for coenzyme A biosynthesis and metabolism. In the present work, the Mtb panD gene was PCR amplified, overexpressed, and purified by metal affinity chromatography. The recombinant Mtb panD was found to exist as a tetramer in solution. Incubation of Mtb panD at 37 degrees C for several hours resulted in a complete cleavage of the inactive (pi) form into the two subunits (alpha and beta). The cleavage was confirmed by Western blot analysis as well as by N-terminal sequencing. Cleaved Mtb panD was assayed for decarboxylase activity with L-aspartate as substrate. The kinetic parameters K(m) and k(cat) were found to be 219 microM and 0.65s(-1), respectively. These results provide the means for further studies based on the identification of the Mtb panD as well as other components of pantothenate metabolism as potential drug targets.