INTRODUCTION:

Continuous efforts are made worldwide to reduce the unnecessary use of antibiotics, thereby to prevent the development of antibiotic resistance. Although several developed countries have brought several acts to restrictthe use of antibiotics, nevertheless antibiotic resistance continues to emerge¹. Many pathogenic microbes developed resistance by adapting to the existing antibiotics and treatment regimes.

Hence, identifying a new antibiotic molecule that devours the antibiotic resistance is one of the major hurdles in the human healthcare. Antibiotics are usually small molecular weight compounds, that specifically inhibit a key functional protein in the pathogenic organisms, resulting in growth inhibition or death of the pathogens². About 70% of the antibiotics available in the market are derived from Streptomyces. Streptomyces are soil-inhabiting, filamentous bacteria, belonging to Actinomycetales, that are responsible for the characteristic earthy odour of soil especially during rain ². Streptomyces is well known for its ability to produce avariety of antibiotics and new chemical entities. Streptomycin is the first antibiotic derived from Streptomyces, by Waksman, effectively used for the treatment of Tuberculosis. It is evident from several reports that, Streptomyces are a valuable source for novel and effective antibiotics.

Computational methods have revolutionized the process of drug discovery by pharma industry and have significantly reduced the time and cost involved in thediscovery of new drugs. Computational tools were extensively used to screen several thousand chemical compounds and to identify the one that can serve as a drug molecule against a specific pathogen. A major portion of the drug discovery process is relied on identifying moleculeswith thedesired bioactivity. In-Silico molecular docking analysis made this process as a very simple method to identify the drug target by protein-ligand docking analysis³. Several pharmaceutical industries and companies are investing over $800 million in establishing in-Silico drug discovery and drug design facilities, which implies the importance and effectiveness of this drug discovery process³. Recent developments in synthetic biology techniques have the potential to further speedupnatural product drug discoveryand would provide ample opportunity for identification natural chemical products as drug leads⁴. Bioprospecting of natural products as antibacterial and antifungal bioactive leads through molecular docking studies are gaining momentum. In this study, theantibioticpotency of secondary metabolites produced by marine Streptomyces sp. VITAK1 was explored through in-Silico protein-ligand docking analysis.

MATERIALS AND METHODS:

Isolation of actinomycetes:

Marine coastal soil samples were obtained from Andaman and Nicobar Islands, India in a sterile polythene bag and transferred to the laboratory. Marine soil (1g) was serially diluted up to 10^-4 dilution and was plated on to Actinomycetes Isolation Agar (AIA) [Hi-Media] by using spread plate technique^5,6. Colonies obtained in this plate are then further sub-cultured repeatedly to obtain pure cultures.

Antibacterial susceptibility test:

Lawn cultures of bacterial pathogens Staphylococcus aureus (ATCC 33591), Pseudomonas aeruginosa (ATCC 27853), Bacillus cereus (ATCC 14579), Shigellaflexneri (ATCC 12022), Salmonellaparatyphi A (ATCC 9150), Klebsiella pneumoniae (ATCC 33495), Shigellaboydii (ATCC 9207), Proteus vulgaris (ATCC 6380) and Escherichia coli (ATCC 25922) were prepared on Muller Hinton agar medium. Wells were punched in the agar medium using sterile well borers. Either the cell-free culture supernatant (CFCS) or crude extract or pure compound is added to these wells, at various concentrations. The plates are incubated over night at 37ºC and observed for thezone of inhibition^7,8.

Fermentation of actinomycetes isolates:

International Streptomyces Project No.1 (ISP No.1) broth (800ml) was inoculated with the selected isolates in a 1L conical flask and incubated at 37ºC for 7 days at 110rpm in arotary shaker. After incubation, CFCS was obtained by filtering the broth using Whatman No.1 filter paper and the filtrate was mixed with equal volume of Petroleum Ether (1:1). The mixture was shaken vigorously for 1 hour. The organic phase was separated using separation flask. The petroleum ether is then concentrated by using rotaryvacuum evaporator ⁹.For the isolate VITAK1, 32 liters of CFCS was extracted with petroleum ether thrice and concentrated using rotatory vacuum evaporator.

Purification of crude extract:

PE extract was separated byethyl acetate: chloroform (8:2 v/v) solvents on TLC plate and the bands were visualized. The PE extract (2 g) was purified using column chromatography using silica gel (60–120 mM mesh size, SRL, India) column(size 300 x 18 mm) and ethyl acetate (CH₃-COO-CH₂-CH₃) and chloroform (CHCl₃) in 8:2 ratio (v/v)was used as solvent mixture. Eluted fractions (135 nos.) each 20 ml volume at a flow rate of 1 ml/minwere collected. The active fraction was further separated using TLC with ethyl acetate (CH₃-COO-CH₂-CH₃) and chloroform (CHCl₃) in 8:2 ratio (v/v) was used asasolvent.

Spectroscopic analysis:

Petroleum ether crude extract of VITAK1 and also the pure compound obtained from VITAK1 was subjected to GC-MS analysis, to identify the components of crude and to identify the mass of the pure compound. The crude and pure compound wasanalyzed in a Perkin Elmer Clarus 680 equipped with Mass spectrometer Clarus 600 (EI) fitted with Elite-5MS capillary column (30m, 0.25mm ID, 250μm df). The GC oven was maintained at the initial temperature of 60°C for 2 min, ramp temperature 10°C/min to 300°C, hold 6min. The temperature of theinjector was 250°C and the oven temperature was maintained at 300°C for 6min. Helium carrier gas was used with a constant flow rate of 1mL/min. Mass transfer line and source temperature were set at 240°C. Turbo Mass version 5.4.2 software were used for the spectral analysis. Structure determination was done by comparison of mass spectral patterns to the NIST-2008 library¹⁰.

The FT-IR spectrum of the pure compound was recorded in Perkin Elmer Spectrum1 FTIR spectrometer at a scan range of 450 to 4000 cm⁻¹, to identify the functional groups present in the pure compound¹¹.¹H NMR, 13C NMR, DEPT-90, DEPT-130 analysis of the pure fraction was analysed using AV500 FT-NMR spectrometer to identify the positioning of hydrogen and carbon¹².

Protein-Ligand docking analysis:

Protein structures were downloaded from RCSB website, with the following PDB ID: 2ZDQ, 3SRW, 2VEG, 3TTZ, 1JZQ, 4UWI, 1E9X and 1FI4 (www.rcsb.org). The ligand molecule was drawn using Marvin sketch tool and was saved in the PDB format for interaction studies. The protein-ligand interaction was studied using AutoDock4.2 in the MGLTools-1.5.6. The results from AutoDock4.2 is then analysed in PyMOL software¹³.

RESULTS:

Screening of actinomycetes isolates:

A total of 12 actinomycetes were isolated with different morphological appearances from Andaman and Nicobar Islands soil samples. All isolates were subjected to antibacterial activity screening through agar well diffusion method, by using the cell-freeculture supernatant (CFCS) of the actinomycetes isolates. Among the 12 isolates, VITAK1 showed potential antibacterial activity was mass cultured for extraction of secondary metabolites.

Morphological characterization:

Morphological studies showed that the isolate VITAK1 produced pinkish white to red in colour colonies, powdery with irregular marginin AIA medium. The aerial mycelium was pinkish white and substrate mycelium was white in color. The morphology if VITAK1 was compared with that ofStreptomyces fradiae (Figure 1). The colony morphology of VITAK1(Figure 1B) was similar toStreptomyces

fradiae(Figure 1A). SEM image of VITAK1 (Figure 1D) was similar to that of Streptomyces fradiae (Figure 1C).

Figure 1: Colony morphology and SEM image of Streptomyces sp. VITAK1and Streptomyces fradiae(reference strain). A) Colony morphology of VITAK1, B) Streptomyces fradiae. C) SEM image of VITAK1 and D) Streptomyces fradiae.

Molecular characterization:

The Genomic DNA of VITAK1 was extracted and 16S rDNA region was amplified using PCR. The PCR product of ~1500 bp is shown in electrophoresis gel (Figure 2A). The sequencing of PCR product of 16S rDNA yielded 1296 nucleotides.16S rRNA partial gene sequence (1296 nucleotides) was submitted to the NCBI database-GenBankunder the accession ID: KF478916. BLAST search of16S rDNA nucleotide sequence of VITAK1 with Gen Bank data base showed 99% homology with Streptomyces fradiae NR0434851. The phylogenetic relationship of VITAK1 with Streptomyces fradiaeis shown in Figure 2B.

Comparison with reference strain:

The physiochemical properties of the isolate VITAK1 have been compared with the identified reference strain Streptomyces fradiae (Table 1). Both the strains had similarities in many of the morphological parameters, they differed in their ability to produce melanin, reverse pigmentation, and substrate mycelium colour.

Table 1: Comparison of physiochemical properties of VITAK1 with the reference strain Streptomyces fradiae

Characteristics	Streptomyces sp. VITAK1	Streptomyces fradiae
Gram strain	+	+
Mobility	-	-
Endospore staining	-	-
Spores	Smooth	Smooth
Colony colour	Pinkish red	Brown
Aerial mycelium	Pinkish white	White
Substrate mycelium	White	Red
Production of pigments	Red	Red
Reverse side pigment	Negative	Yellow red
Melanin production	Negative	Positive
Starch hydrolysis	Negative	Not Defined
NaCl	2%	2%
Temperature	30 °C	28 °C

Antibacterial activity of VITAK1:

The isolate VITAK1was mass cultured in ISP No.1 (International Streptomyces Project Number 1) broth media and the CFCS was extracted with petroleum ether. The crude petroleum ether extract was analyzed for its antibacterial activity against 9 bacterial pathogens at a concentration of 100µg/ml. The extract demonstrated the highest zone of inhibition of 22±1.04mm against Staphylococcus aureus and lowest zone of inhibition against Bacillus cereus (Table 2). Compared to the standard 10µg of ciprofloxin, the crude extract demonstrated higher activity.

Bioactivity-guided purification of crude extract:

The PE extract of culture brothyielded a crude compound 0.055 g/L. TLC separation of PE extract with ethyl acetate: chloroform (8:2 v/v) solvent mixture showed four bands. 2 g of PE extract was subjected to column chromatographic separation. All the fractions were subjected to antibacterial activity screening and fractions 25 to78 (brown coloured) showed antibacterial activity wascollected, concentrated and lyophilized. The active fraction yield was calculated to be 350 mg and it was further purified using preparative –HPLC. Three peaks were found in the preparative-HPLC chromatogram, a major peak at 3.079 min and two minor peaks at 5.497 and 8.82min respectively. HPLC chromatogramis shown in Figure 3 suggests that the major peak has 99% of the major compound (compound 1) and 1% of impurities (compound 2 and 3). The purified fractions were screened for antibacterial activity and found to have antibacterial activityagainst tested bacterial pathogens. TLC separation of active fraction yielded a distinct single band (R_f: 0.67). Minor peaks are not shown any antibacterial activity. The lead compound was lyophilized and subjected to further characterization.

Structure elucidation of compound 1:

The purified compound 1 was subjected to various spectroscopic analysis such as; FTIR, GCMS, ¹H NMR, ¹³C NMR, DEPT90, and DEPT135. Based on the interpretation ofspectral data obtained from the spectroscopic analysis, the structure of the pure compound was identified to be (3E, 5E, 7E, 9E, 11E, 13E,15E)-16-(pyrrolidin-1-yl) hexadeca-3, 5, 7, 9, 11, 13, 15-heptaen-2-one with a molecular weight of 295.41 Da and molecular formula C₂₀H₂₅NO.

The ¹HNMR shifts are shown in Figure 4. The individual methyl group appears as a single shift, since, there are no neighbouring hydrogen atoms. The two carbons in the pyrrolo ring produce a pentaplet, since, they are isomeric atoms, both produce the same shift, resulting in one pentaplet. The remaining atoms in the molecules, however, produce only a triplet, since all the atoms are having two neighbouring hydrogen atoms.

The ¹³C NMRspectrum is shown in Figure 5. ¹³C NMR spectrum showed the presence of 19 carbon atoms. But, the carbons present in the Pyrrol group did not produce individual peaks, resulting in loss of a carbon shift in the NMR spectrum. Hence, in the spectrum, it was observed only 19 shifts, instead of 20 shifts.

The phase shifts in the DEPT-90 analysis is shown in Figure 6. It confirmed the presence of 14*(-C-H) group in the compound. The total number of (-C-H) group in the compound is fourteen and has been highlighted in Figure 6.

The phase shifts intheDEPT-135 analysis is shown in Figure 7. It confirmed the presence of 4* (-CH₂) group in the compound. Four negative peaks, signifying the (-CH₂) group are highlighted in Figure 7.Mass spectrum revealed the molecular weight of the compound as 295.39 Da (Figure 8). The exact mass of the interpreted structure is 295.41 Da. The mass spectrum data was validated.

The presence of (-C=O) stretching at 1743cm^-1indicates the presence of ketone group and stretching at 1180cm^-1 indicate the presence of (-C-N-) amine group (Figure 9). FT-IR data was validated with the interpreted structure.

Interpretation of spectral data results in identification of the structure of the pure compoundand identified as (3E, 5E, 7E, 9E, 11E, 13E, 15E)-16-(pyrrolidin-1-yl) hexadeca-3, 5, 7, 9, 11, 13, 15-heptaen-2-one (Figure 10), also called as pyrrolidinyl-hexadeca-heptaenone (PHDH).

Figure 4.¹HNMR spectrum of the pure compound from VITAK1

Figure 5: ¹³C NMR spectrum of the pure compound from VITAK1

Figure 6: DEPT-90 NMR analysis of the pure compound from VITAK1

Figure 7: DEPT-135 NMR analysis of the pure compound from VITAK1

Figure 10. Structure of the pure compound pyrrolidinyl-hexadeca-heptaenonefrom VITAK1

In Silico Protein-Ligand docking:

The lead compound PHDH was analysedin-Silico for its ability to inhibit known antifungal and antibacterial proteins. The interaction of PHDH with antibacterial drug targets and their binding energy is given in Table 3.Among the 5 bacterial protein targets analysed, PHDH demonstrated thehighest affinity towards dihydropteroate synthase enzyme with the least binding energy of -8.14 K cal/mol. This is a key enzyme involved in thebiosynthesis of folate and based on docking results, it is proposed that PHDH exhibits antibacterial activity, by inhibiting the folate synthesis.

The interaction of PHDH with antifungal drug targets and their binding energy is given in Table 4. PHDH demonstrated high-affinity interaction with myristoyltransferaseprotein, which is a key enzyme involved in ergosterol production. Basedon docking studies, PHDH exhibits antifungal activity by inhibiting myristoyltransferase protein.

Table 3: Interaction of PHDH with antibacterial drug targets

Protein Name	Binding Energy (K cal/mol)	Inhibition Constant (μM)
DihydrofolateReductase	-8.11	1.13
DihydropteroateSynthase	-8.14	1.07
D-Alanine Ligase	-1.57	70.45
Topoisomerase	-5.51	92.06
TRNASynthase	-4.6	424.38

Table 4: Interaction of PHDH with antifungal drug targets

Protein Name	Binding Energy (K cal/mol)	Inhibition Constant (μM)
14aDemthylase	-7.22	5.08
M5DD	-6.51	16.84
MyristoylTransferase	-8.8	353.21

DISCUSSION:

Currently, protein-ligand docking studies arewidely used for drug-receptor interaction analysis. Protein-Liganddocking provides more information on drug-protein interactions and it is being usedmore often to predict the exact binding orientation of drug molecules (ligands) with their targets to ascertain the affinity and interaction ofligands ¹⁴. Novel and effective antibiotic molecules are in urgent need to combatthe increased incidence of drug resistance. Antimicrobial activity of several secondary metabolites derived from actinomycetes was predicted by molecular docking studies which include, theantifungal activity of thelead molecule, 5-(2, 4-dimethylbenzyl) pyrrolidin-2-one (DMBPO) derived from a novel Streptomyces sp.VITSVK5against fungal drug targets¹⁵; theantifungal activity of ester compoundsderivedfrom Streptomyces sp. VITSTK7 ¹⁶; antibacterial activity of compound, 2, 5-di-tert-butyl-1, 4-benzoquinone (DTBBQ) derived from Streptomyces VITVSK1 spp.¹⁷; antibacterial activity and preclinical evaluation of 2,5-di-tert-butyl-1,4-benzoquinone (DTBBQ) from Streptomyces sp. VITVSK1¹⁸.

Several novel and effective antibiotic molecules produced by marine actinomycetes have been reported, which include, 1, 2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester produced by Streptomyces sp VITSJK8 demonstrated antibacterial activity against ESBL pathogens with an MIC₅₀ value of 0.13 to 2.0 µg/ml^[7].Two antibacterial secondary metabolites, 3-ethyl,3-methyl heptanes and diisodecyl ether were identified from Streptomyces coelicolorstrain SU6 a marine Streptomyces isolate that demonstrated strong antibacterial activity against common human pathogens¹⁹. Rhodomycin-B is a novel secondary metabolite produced by Streptomyces purpurascensa marine actinomycetes strain, demonstrated a significant antibacterial activity with MIC value of 2µg/ml against Bacillus subtilis²⁰.

Streptomyces sp. VITAK1 also produced another antibacterial secondary metabolite, coumarin-6ol,3,4-dihydro-4,4,5,7-tetra methyl (CDTM)^[21]. It showed significant antibacterial activity againstGram-positive and Gram negative bacterial pathogens.It showed the MIC₅₀ value of 2.5µg/ml against Proteus vulgaris²². CDTM showed thehighest affinity and the least binding free energy of -8.12 Kcal/molwith bacterial drug target protein D-alanine:D-alanine ligase (PDB ID: 2ZDQ). During the interaction,it formed 2 H bonds, Glu-197 (2.0 Å) and Tyr-223 (2.1 Å) with the inhibition constant (Ki) 1.12 μM²³.Several other studies have reported such potential antibacterial activity of actinomycetes isolates ²⁴.

CONCLUSION:

Streptomyces sp.VITAK1 derived secondary metabolite PHDH is a novel compound capable of inhibiting fungal enzymemyristoyltransferase and bacterial protein dihydropteroate synthase. PHDH can be studied further for its interaction with other bacterial and fungal drug targetsboth by in vitro and in vivo studies to establish its broad spectrum of activity. The antibiotic potential of the novel compound PHDH can be studied further to develop as adrug.

ACKNOWLEDGMENTS:

The authors thank the managementofVIT Universityfor providing facilities to carry out this study.

CONFLICT OF INTEREST:

The authors declare no conflict of interest

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Received on 28.06.2017 Modified on 18.07.2017

Research J. Pharm. and Tech 2018; 11(5):1901-1908.

DOI: 10.5958/0974-360X.2018.00353.0

Bacterial pathogens	VITAK1 Zone of inhibition (mm) (Crude extract, 100 µg/ml)	Ciprofloxin (10 µg/ml)
Staphylococcus aureus (ATCC 33591)	22±1.04	14±1.09
Bacillus cereus (ATCC 14579)	19.5±0.5	14±0.46
Salmonella paratyphiA (ATCC 9150)	19.66±1.52	14±0.37
Klebsiellapneumoniae(ATCC 33495)	12.16±1.04	16±0.28
Pseudomonas aeruginosa (ATCC 27853)	12.83±0.28	17±0.16
Shigellaflexneri(ATCC 12022)	12.2±0.28	14±0.25
Shigellaboydii(ATCC 9207)	12.0±0.5	14±0.28
Proteus vulgaris (ATCC 6380)	9.83±0.29	18±0.87
Escherichia coli (ATCC 25922)	12.2±0.76	19±0.54