Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

Identification of diphenylurea derivatives as novel endocytosis inhibitors that demonstrate broad-spectrum activity against SARS-CoV-2 and influenza A virus both in vitro and in vivo.

Rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) poses enormous challenge in the development of broad-spectrum antivirals that are effective against the existing and emerging viral strains. Virus entry through endocytosis represents an attractive target for drug development, as inhibition of this early infection step should block downstream infection processes, and potentially inhibit viruses sharing the same entry route. In this study, we report the identification of 1,3-diphenylurea (DPU) derivatives (DPUDs) as a new class of endocytosis inhibitors, which broadly restricted entry and replication of several SARS-CoV-2 and IAV strains. Importantly, the DPUDs did not induce any significant cytotoxicity at concentrations effective against the viral infections. Examining the uptake of cargoes specific to different endocytic pathways, we found that DPUDs majorly affected clathrin-mediated endocytosis, which both SARS-CoV-2 and IAV utilize for cellular entry. In the DPUD-treated cells, although virus binding on the cell surface was unaffected, internalization of both the viruses was drastically reduced. Since compounds similar to the DPUDs were previously reported to transport anions including chloride (Cl-) across lipid membrane and since intracellular Cl- concentration plays a critical role in regulating vesicular trafficking, we hypothesized that the observed defect in endocytosis by the DPUDs could be due to altered Cl- gradient across the cell membrane. Using in vitro assays we demonstrated that the DPUDs transported Cl- into the cell and led to intracellular Cl- accumulation, which possibly affected the endocytic machinery by perturbing intracellular Cl- homeostasis. Finally, we tested the DPUDs in mice challenged with IAV and mouse-adapted SARS-CoV-2 (MA 10). Treatment of the infected mice with the DPUDs led to remarkable body weight recovery, improved survival and significantly reduced lung viral load, highlighting their potential for development as broad-spectrum antivirals.

Brevicillin, a novel lanthipeptide from the genus Brevibacillus with antimicrobial, antifungal, and antiviral activity.

AIM: This study was aimed to determine antimicrobial and antiviral activity of a novel lanthipeptide from a Brevibacillus sp. for disinfectant application. METHODS AND RESULTS: The antimicrobial peptide (AMP) was produced by a bacterial strain AF8 identified as a member of the genus Brevibacillus representing a novel species. Whole genome sequence analysis using BAGEL identified a putative complete biosynthetic gene cluster involved in lanthipeptide synthesis. The deduced amino acid sequence of lanthipeptide named as brevicillin, showed >30% similarity with epidermin. Mass determined by MALDI-MS and Q-TOF suggested posttranslational modifications like dehydration of all Ser and Thr amino acids to yield Dha and Dhb, respectively. Amino acid composition determined upon acid hydrolysis is in agreement with core peptide sequence deduced from the putative biosynthetic gene bvrAF8. Biochemical evidence along with stability features ascertained posttranslational modifications during formation of the core peptide. The peptide showed strong activity with 99% killing of pathogens at 12 µg ml-1 within 1 minute. Interestingly, it also showed potent anti-SARS-CoV-2 activity by inhibiting ∼99% virus growth at 10 µg ml-1 in cell culture-based assay. Brevicillin did not show dermal allergic reactions in BALB/c mice. CONCLUSION: This study provides detailed description of a novel lanthipeptide and demonstrates its effective antibacterial, antifungal and anti-SARS-CoV-2 activity.

Structural characterization of Myxococcus xanthus MglC, a component of the polarity control system, and its interactions with its paralog MglB.

The δ-proteobacteria Myxococcus xanthus displays social (S) and adventurous (A) motilities, which require pole-to-pole reversal of the motility regulator proteins. Mutual gliding motility protein C (MglC), a paralog of GTPase-activating protein Mutual gliding motility protein B (MglB), is a member of the polarity module involved in regulating motility. However, little is known about the structure and function of MglC. Here, we determined ∼1.85 Å resolution crystal structure of MglC using Selenomethionine Single-wavelength anomalous diffraction. The crystal structure revealed that, despite sharing <9% sequence identity, both MglB and MglC adopt a Regulatory Light Chain 7 family fold. However, MglC has a distinct ∼30° to 40° shift in the orientation of the functionally important α2 helix compared with other structural homologs. Using isothermal titration calorimetry and size-exclusion chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using small-angle X-ray scattering and molecular docking studies, we show that the MglBC complex consists of a MglC homodimer sandwiched between two homodimers of MglB. A combination of size-exclusion chromatography and site-directed mutagenesis studies confirmed the MglBC interacting interface obtained by molecular docking studies. Finally, we show that the C-terminal region of MglB, crucial for binding its established partner MglA, is not required for binding MglC. These studies suggest that the MglB uses distinct interfaces to bind MglA and MglC. Based on these data, we propose a model suggesting a new role for MglC in polarity reversal in M. xanthus.

Disruption of MenT2 toxin impairs the growth of Mycobacterium tuberculosis in guinea pigs.

Toxin-antitoxin (TA) systems are abundantly present in the genomes of various bacterial pathogens. TA systems have been implicated in either plasmid maintenance or protection against phage infection, stress adaptation or disease pathogenesis. The genome of Mycobacterium tuberculosis encodes for more than 90 TA systems and 4 of these belong to the type IV subfamily (MenAT family). The toxins and antitoxins belonging to type IV TA systems share sequence homology with the AbiEii family of nucleotidyl transferases and the AbiEi family of putative transcriptional regulators, respectively. Here, we have performed experiments to understand the role of MenT2, a toxin from the type IV TA system, in mycobacterial physiology and disease pathogenesis. The ectopic expression of MenT2 using inducible vectors does not inhibit bacterial growth in liquid cultures. Bioinformatic and molecular modelling analysis suggested that the M. tuberculosis genome has an alternative start site upstream of the annotated menT2 gene. The overexpression of the reannotated MenT2 resulted in moderate growth inhibition of Mycobacterium smegmatis. We show that both menT2 and menA2 transcript levels are increased when M. tuberculosis is exposed to nitrosative stress, in vitro. When compared to the survival of the wild-type and the complemented strain, the ΔmenT2 mutant strain of M. tuberculosis was more resistant to being killed by nitrosative stress. However, the survival of both the ΔmenT2 mutant and the wild-type strain was similar in macrophages and when exposed to other stress conditions. Here, we show that MenT2 is required for the establishment of disease in guinea pigs. Gross pathology and histopathology analysis of lung tissues from guinea pigs infected with the ∆menT2 strain revealed significantly reduced tissue damage and inflammation. In summary, these results provide new insights into the role of MenT2 in mycobacterial pathogenesis.

Antifungal Azoles as Tetracycline Resistance Modifiers in Staphylococcus aureus.

Staphylococcus aureus has developed resistance to antimicrobials since their first use. The S. aureus major facilitator superfamily (MFS) efflux pump Tet(K) contributes to resistance to tetracyclines. The efflux pump diminishes antibiotic accumulation, and biofilm hampers the diffusion of antibiotics. None of the currently known compounds have been approved as efflux pump inhibitors (EPIs) for clinical use. In the current study, we screened clinically approved drugs for possible Tet(K) efflux pump inhibition. By performing in silico docking followed by in vitro checkerboard assays, we identified five azoles (the fungal ergosterol synthesis inhibitors) showing putative EPI-like potential with a fractional inhibitory concentration index of ≤0.5, indicating synergism. The functionality of the azoles was confirmed using ethidium bromide (EtBr) accumulation and efflux inhibition assays. In time-kill kinetics, the combination treatment with butoconazole engendered a marked increase in the bactericidal capacity of tetracycline. When assessing the off-target effects of the azoles, we observed no disruption of bacterial membrane permeability and polarization. Finally, the combination of azoles with tetracycline led to a significant eradication of preformed mature biofilms. This study demonstrates that azoles can be repurposed as putative Tet(K) EPIs and to reduce biofilm formation at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus uses efflux pumps to transport antibiotics out of the cell and thus increases the dosage at which it endures antibiotics. Also, efflux pumps play a role in biofilm formation by the excretion of extracellular matrix molecules. One way to combat these pathogens may be to reduce the activity of efflux pumps and thereby increase pathogen sensitivity to existing antibiotics. We describe the in silico-based screen of clinically approved drugs that identified antifungal azoles inhibiting Tet(K), a pump that belongs to the major facilitator superfamily, and showed that these compounds bind to and block the activity of the Tet(K) pump. Azoles enhanced the susceptibility of tetracycline against S. aureus and its methicillin-resistant strains. The combination of azoles with tetracycline led to a significant reduction in preformed biofilms. Repurposing approved drugs may help solve the classical toxicity issues related to efflux pump inhibitors.

System-Wide Analysis Unravels the Differential Regulation and In Vivo Essentiality of Virulence-Associated Proteins B and C Toxin-Antitoxin Systems of Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems are bicistronic genetic modules that are ubiquitously present in bacterial genomes. The Mycobacterium tuberculosis genome encodes 90 putative TA systems, and these are considered to be associated with maintenance of bacterial genomic stability or bacterial survival under unfavorable environmental conditions. The majority of these in M. tuberculosis have been annotated as belonging to the virulence-associated protein B and C (VapBC) family. However, their precise role in bacterial physiology has not been elucidated. Here, we functionally characterized VapC toxins from M. tuberculosis and show that overexpression of some homologs inhibits growth of Mycobacterium bovis bacillus Calmette-Guérin in a bacteriostatic manner. Expression profiling of messenger RNA revealed that these VapC toxins were differentially induced upon exposure of M. tuberculosis to stress conditions. We also unraveled that transcriptional cross-activation exists between TA systems in M. tuberculosis. This study provides the first evidence for the essentiality of VapBC3 and VapBC4 systems in M. tuberculosis virulence.

Crystal structure of the N-terminal domain of human SIRT7 reveals a three-helical domain architecture.

Human SIRT7 is an NAD(+) dependent deacetylase, which belongs to sirtuin family of proteins. SIRT7, like other sirtuins has conserved catalytic domain and is flanked by N- and C-terminal domains reported to play vital functional roles. Here, we report the crystal structure of the N-terminal domain of human SIRT7 (SIRT7(NTD) ) at 2.3 Å resolution as MBP-SIRT7(NTD) fusion protein. SIRT7(NTD) adopts three-helical domain architecture and comparative structural analyses suggest similarities to some DNA binding motifs and transcription regulators. We also report here the importance of N- and C-terminal domains in soluble expression of SIRT7. Proteins 2016; 84:1558-1563. © 2016 Wiley Periodicals, Inc.

Mycobacterium tuberculosis keto-mycolic acid and macrophage nuclear receptor TR4 modulate foamy biogenesis in granulomas: a case of a heterologous and noncanonical ligand-receptor pair.

The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA-TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

Crystal structure of Mycobacterium tuberculosis CarD, an essential RNA polymerase binding protein, reveals a quasidomain-swapped dimeric structural architecture.

Mycobacterium tuberculosis (Mtb) CarD is an essential transcriptional regulator that binds RNA polymerase and plays an important role in reprogramming transcription machinery under diverse stress conditions. Here, we report the crystal structure of CarD at 2.3 Å resolution, that represents the first structural description of CarD/CdnL-Like family of proteins. CarD adopts an overall bi-lobed structural architecture where N-terminal domain resembles 'tudor-like' domain and C-terminal domain adopts a novel five helical fold that lacks the predicted leucine zipper structural motif. The structure reveals dimeric state of CarD resulting from ß-strand swapping between the N-terminal domains of each individual subunits. The structure provides crucial insights into the possible mode(s) of CarD/RNAP interactions.

Structural basis for the redox sensitivity of the Mycobacterium tuberculosis SigK-RskA σ-anti-σ complex.

The host-pathogen interactions in Mycobacterium tuberculosis infection are significantly influenced by redox stimuli and alterations in the levels of secreted antigens. The extracytoplasmic function (ECF) σ factor σ(K) governs the transcription of the serodominant antigens MPT70 and MPT83. The cellular levels of σ(K) are regulated by the membrane-associated anti-σ(K) (RskA) that localizes σ(K) in an inactive complex. The crystal structure of M. tuberculosis σ(K) in complex with the cytosolic domain of RskA (RskAcyto) revealed a disulfide bridge in the -35 promoter-interaction region of σ(K). Biochemical experiments reveal that the redox potential of the disulfide-forming cysteines in σ(K) is consistent with its role as a sensor. The disulfide bond in σ(K) influences the stability of the σ(K)-RskAcyto complex but does not interfere with σ(K)-promoter DNA interactions. It is noted that these disulfide-forming cysteines are conserved across homologues, suggesting that this could be a general mechanism for redox-sensitive transcription regulation.

Biochemical and structural characterization reveals Rv3400 codes for ß-phosphoglucomutase in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or ß-phosphoglucomutase (ß-PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs ß-PGM activity and hence, Rv3400 encodes for ß-PGM in Mtb. Our data also confirm that Mtb ß-PGM is a metal dependent enzyme having broad specificity for divalent metal ions. ß-PGM converts ß-D-glucose-1-phosphate to ß-D-glucose-6-phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of ß-PGM in the physiology of Mtb.

Mycobacterium tuberculosis strain with deletions in menT3 and menT4 is attenuated and confers protection in mice and guinea pigs.

The genome of Mycobacterium tuberculosis encodes for a large repertoire of toxin-antitoxin systems. In the present study, MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems have been functionally characterized. We demonstrate that ectopic expression of these toxins inhibits bacterial growth and this is rescued upon co-expression of their cognate antitoxins. Here, we show that simultaneous deletion of menT3 and menT4 results in enhanced susceptibility of M. tuberculosis upon exposure to oxidative stress and attenuated growth in guinea pigs and mice. We observed reduced expression of transcripts encoding for proteins that are essential or required for intracellular growth in mid-log phase cultures of ΔmenT4ΔT3 compared to parental strain. Further, the transcript levels of proteins involved in efficient bacterial clearance were increased in lung tissues of ΔmenT4ΔT3 infected mice relative to parental strain infected mice. We show that immunization of mice and guinea pigs with ΔmenT4ΔT3 confers significant protection against M. tuberculosis infection. Remarkably, immunization of mice with ΔmenT4ΔT3 results in increased antigen-specific TH1 bias and activated memory T cell response. We conclude that MenT3 and MenT4 are important for M. tuberculosis pathogenicity and strains lacking menT3 and menT4 have the potential to be explored further as vaccine candidates.

Molecular docking and dynamic simulation approach to decipher steroidal sapogenins (genus Trillium) derived agonists for glucocorticoid receptor.

Steroidal sapogenins (SS) are structural analogues of steroidal drugs, which are frequently used for the treatment of several diseases including reproductive, malignancies, neurological, and inflammation-related diseases. The glucocorticoid receptor (GR) is a nuclear receptor that regulates development, metabolism, and inflammation, in response to steroidal ligands. Therefore, GR is considered as a potential therapeutic target for steroidal agents to the treatment of inflammation-related diseases. We hypothesized that SS may act as an agonist for GR due to structural similarity with corticosteroids. In this study, we carried out in silico screening of various SS from the genus Trillium to check their potential as an agonist for GR. Our data suggest that out of 42 SS, only 7 molecules have interacted with GR. However, molecular mechanics with generalized Born and surface area (MM-GBSA) analysis revealed that only two SS (SS 38 and SS 39) molecules bind favorably to GR. Among these, SS 38 (docking score: -9.722 Kcal/mol and MM-GBSA ΔGbind: -50.192 Kcal/mol) and SS 39 (docking score: -11.20 Kcal/mol and MM-GBSA ΔGbind: -58.937 Kcal/mol) have best docking and MM-GBSA scores. Molecular dynamics (MD) simulation studies of SS 38, SS 39, and dexamethasone-GR complex revealed that both SS shows hydrogen bonding and hydrophobic interaction with GR over the 120 ns simulation with mild fluctuations. The current study suggests that SS 38 and SS 39 may be further explored as a potential agonist to treat several disease conditions mediated by GR.

Structural insights into DarT toxin neutralization by cognate DarG antitoxin: ssDNA mimicry by DarG C-terminal domain keeps the DarT toxin inhibited.

In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2 Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions.

Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes.

The phenomenon of protein aggregation is associated with a wide range of human diseases. Our knowledge of the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated this behaviour in the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions (APRs) in these proteomes and revealed a strong aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone SARS-CoV and SARS-CoV-2 proteins and protein regions, including the signal sequence peptide and fusion peptides 1 and 2 of the spike protein, a peptide from the NSP6 protein, and the ORF10 and NSP11 proteins. Our results show that these peptides and proteins can form amyloid aggregates. We used circular dichroism spectroscopy to reveal the presence of ß-sheet rich cores in aggregates and X-ray diffraction and Raman spectroscopy to confirm the formation of amyloid structures. Furthermore, we demonstrated that SARS-CoV-2 NSP11 aggregates are toxic to mammalian cell cultures. These results motivate further studies about the possible role of aggregation of SARS proteins in protein misfolding diseases and other human conditions.

UVC-Based Air Disinfection Systems for Rapid Inactivation of SARS-CoV-2 Present in the Air.

The World Health Organization (WHO) declared in May 2021 that SARS-CoV-2 is transmitted not only by close contact with infectious respiratory fluids from infected people or contaminated materials but also indirectly through air. Airborne transmission has serious implications for the control measures we can deploy, given the emergence of more transmissible variants. This emphasizes the need to deploy a mechanism to reduce the viral load in the air, especially in closed and crowded places such as hospitals, public transport buses, etc. In this study, we explored ultraviolet C (UVC) radiation for its ability to inactivate the SARS-CoV-2 particles present in aerosols and designed an air disinfection system to eliminate infectious viruses. We studied the virus inactivation kinetics to identify the UVC dosage required to achieve maximum virus inactivation. Based on the experimental data, UVC-based devices were designed for the sanitization of air through HVAC systems in closed spaces. Further, a risk assessment model to estimate the risk reduction was applied which showed that the use of UVC radiation could result in the reduction of the risk of infection in occupied spaces by up to 90%.

Potential Inhibitors of SARS-CoV-2 Main Protease (Mpro) Identified from the Library of FDA-Approved Drugs Using Molecular Docking Studies.

The Corona Virus Infectious Disease-2019 (COVID-19) outbreak originated at Wuhan, China, in December 2019. It has already spread rapidly and caused more than 6.5 million deaths worldwide. Its causal agent is a beta-coronavirus named SARS-CoV-2. Many efforts have already been made to develop new vaccines and drugs against these viruses, but over time, it has changed its molecular nature and evolved into more lethal variants, such as Delta and Omicron. These will lead us to target its more-conserved proteins. The sequences' BLAST and crystal structure of the main protease Mpro suggest a high sequence and structural conservation. Mpro is responsible for the proteolytic maturation of the polyprotein essential for the viral replication and transcription, which makes it an important drug target. Discovery of new drug molecules may take years before getting to the clinics. So, considering urgency, we performed molecular docking studies using FDA-approved drugs to identify molecules that could potentially bind to the substrate-binding site and inhibit SARS-CoV-2's main protease (Mpro). We used the Glide module in the Schrödinger software suite to perform molecular docking studies, followed by MM-GBSA-based energy calculations to score the hit molecules. Molecular docking and manual analysis suggest that several drugs may bind and potentially inhibit Mpro. We also performed molecular simulations studies for selected compounds to evaluate protein-drug interactions. Considering bioavailability, lesser toxicity, and route of administration, some of the top-ranked drugs, including lumefantrine (antimalarial), dipyridamole (coronary vasodilator), dihydroergotamine (used for treating migraine), hexoprenaline (anti asthmatic), riboflavin (vitamin B2), and pantethine (vitamin B5) may be taken forward for further in vitro and in vivo experiments to investigate their therapeutic potential.