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Fundamentals of Drug Discovery

Fundamentals of Drug Discovery

Practical Report of Lab Work

  • Screening of secondary metabolites from Actinomyces
  • Strains Collection and Cultivation
  • Extraction of active compounds
  • Biological screening
  • Chemical Screening
  • Column Chromatography
  • Preparative TLC
  • Metagenomic DNA Isolation

Fundamentals of Drug Discovery – Introduction:

Organic compounds from terrestrial and marine organisms have extensive past and present use in the treatment of many diseases and serve as compounds of interest both in their natural form and as templates for synthetic modification. Over 20 new drugs launched on the market between 2000 and 2005, originating from terrestrial plants, terrestrial microorganisms, marine organisms, and terrestrial vertebrates and invertebrates, are described. These approved substances, representative of very wide chemical diversity, together with several other natural products or their analogs undergoing clinical trials, continue to demonstrate the importance of compounds from natural sources in modern drug discovery efforts.For thousands of years, natural products have played an important role throughout the world in treating and preventing human diseases. Natural product medicines have come from various source materials including terrestrial plants, terrestrial microorganisms, marine organisms, and terrestrial vertebrates and invertebrates. The importance of natural products in modern medicine has been discussed in recent reviews and reports. The value of natural products in this regard can be assessed using 3 criteria: the rate of introduction of new chemical entities of wide structural diversity, including serving as templates for semisynthetic and total synthetic modification, the number of diseases treated or prevented by these substances, and their frequency of use in the treatment of disease.

The integrated Approach of Antibiotics Screening are:Microbiology: it includes Isolation of Bacteria, characterization, phylogenetic studies, culture preservation, Biological Screening (antimicrobial activities, cytotoxic studies etc).Medicine: it includes Clinical trials, cell line inhibition etc. Biotechnology: it is the Genomics, Scale up, media optimization, strain improvement, Fermentation etc. Chemistry: it includes Chemical screening, isolation and purification of compounds, structure elucidation etc.

Isolation and characterization of the target organism consist of two screening procedures:

Prescreening screening: (biological and chemical screening , selection of competent strains)Culture preservation Cultural optimizations

Preparative Screening: (Fermentation, isolation purification and structure elucidation of the metabolitesAssociated genetic and mutational studies

Success Rate in discovery of new therapeutic compound depends on two factors

  • Selection of appropriate strains (30-40% influence)
  • Selection of appropriate test systems (60-70% influence)

The difference between primary screening and Secondary Screening is given below:

Primary bioassays

  • Can be rapidly applied to a large number of samples
  • Should have high capacity, low cost and provide the results quickly
  • Need not be quantitative

Secondary testing

  • involve more detailed testing of lead compounds to select compounds for clinical trials
  • usually low capacity, slow and costly assays

After selection of source which is targeted for obtaining active compound. The active compound is extracted by solvent extraction methods. After extraction of active compound the biological screening (antimicrobial activity and cytotoxicity assay) and chemical analysis of active compound is done by chemical screening that (include HPLC and TLC). The column chromatography is used for the purification of fractions of active compound.

Fig1 : Solvent extraction by separating funnal method           Fig2 : Celite Bed preparation and extraction

Biological Screening Results

In-vitro Inhibition of (gram –ve)and (gram +ve) bacteria by natural extracts by Disc diffusion Method

Results of disc diffusion are given below:

 

Extracts name

Zone of inthibition on Test strains(mm)

Gram positive bacteria

Gram negative bacteria

AS-1

5

5.2

S1-D

5.1

D-9

6

5.3

D-11

6.2

5.9

emp

St

5.9

 Cytotoxicity Assay Results:

Artemia Salina (brine shrimp) has been attributed as a model organism for laboratories to determine the toxicological effects of chemicals used in biological assays. Artemiasalina is easy to breed in laboratories and is inexpensive. Its dried eggs are available to hatch them in artificial sea water that is continuously aerated.

Cytotoxicity results

S. No

Strain

G

N

A

M(% mortality)

1

D-11

23

2

11

28

2

Q2-12

21

1

14

50

3

Q2-A

25

5

17

54

4

D-16

20

1

9

26

5

S1

21

1

10

30

6

Blank

25

2

1

0

CHEMICAL SCREENING (Metabolic profile of the selected Actinomyces strains)

Thin Layer Chromatography (TLC)

The crude extracts of strains were analyzed by thin layer chromatography using Anisaldehyde/H2SO4 and Ehrlich’s reagent solution as staining reagents. TLC developed plate was first viewed under short UV wavelength (254nm). The results are given below:

A                                             B

 

C                                                 D

Figure :Chemical screening using TLC of strain AS-1, S1-D, D-9, andD-11

A = TLC plate under UV at short wavelength (254 nm)

B = TLC plate under UV at long wavelength (366 nm)

C = TLC plate after treatment with Anisaldehye / H2SO4

D = TLC plate after treatment with Ehrlisch reagent

 

High Performance Liquid Chromatography (HPLC) analysis results:

Figure :HPLC-UV chromatogram of Streptomyces of strain AS-

As-1 shows one major peak at 2.47min.

Discussion:

For the discovery of new active compound from natural sources all of these procedures provide an active attempt that how we cultivate the targeted strain and how we isolate active compound from source (strain) and how we screen them for their biological activity and chemical activity. We used GYM media because it is selective media for cultivation of Actinomyces. After growth of strain the active compound extracted from broth media by solvent extraction method by two procedures. As both procedure of solvent extraction methods are effective, but if quantity of broth is large than bed filtration method is more effective as compared to separating funnel method. After effective extraction of active compound the compound is further screened either it shows some biological activity or not. There are various methods which can be used for analysis of antimicrobial activity but we preferred Disc diffusion method because it is quantitative method in which standardize inoculum of test bacteria is used. The results of disc diffusion method show that crude compound is not too effective against gram positive and negative bacteria because small zone of inhibition was obtained.AS-1gave 5mm and 5.2mm zones against gram positive and gram negative bacteria, S1-Dgave 5.1mm zone against gram positive bacteria,D-9gave 6mm and 5.3mm against gram positive and gram negative bacteria, andD-11strain gave 6.2 and 5.9mm zones against gram positive and gram negative bacteria.

The crude extracts were further analyzed for its cytotoxicity activity against Artemia Salina (brine shrimp). The results show that strain Q2-1 has highest cytotoxicity activity 54% as compared to other strain so this cannot be further developed as antibiotic but can be developed as antitumor compound if it has selective cytotoxicity against tumor cells. The loweswt cytotoxicity was given by D-16 strain and it was 26%. The strains with less cytotoxicity can be further develop as antibiotic. The metabolic finger printing of active compound was done by HPLC and TLC. The purification of fractions of crude compound was done by manual column chromatography and preparative TLC. 

Metagenomic DNA isolation

An improved method for purification of intact metagenomic DNA from soil has been developed using Q-Sepharose, which purified the DNA from phenolic and humic acid contaminants in a single step. The entire procedure for purification took only 45 min. The purified DNA readily digested with restriction enzymes and can be further used for molecular applications.

Material and methods

Extraction of metagenomic:

Soil samples was collected and DNA from soil extracted by the method of Zhou et al. (1996). Sieved fine soil (0.5 g) extracted with 1.3 ml of extraction buffer (100 mMTrisCl, pH 8.0, 100 mM EDTA, pH 8.0, 1.5 M NaCl, 100 mM sodium phosphate, pH 8.0, 1% CTAB). After proper mixing, 13 ll of proteinase K (10 mg/ml) was added. All eppendorf tubes were incubated horizontally at 37C with shaking for 45 min, after that 160 ll of 20% SDS was added and vortexed for 30 sec with further incubation at 60C for 2 hours. The sample in each eppendorf was mixed thoroughly after every 15 min interval. The samples were centrifuged at 5000 · g for 10 min. The supernatant was transferred into new eppendorf tubes. The remaining soil pellets were treated three times with 400 ll of extraction buffer, 60 ll of SDS (20%) and kept at 60C for 15 min with intermittent shaking after every 5 min. The supernatants collected from all four extractions was mixed with equal quantity of chloroform and isoamyl alcohol (24:1) and extracted three to four times. Aqueous layer was separated and precipitated with 0.6 volume of isopropanol. After centrifugation at 12,000 · g for 15 min, the brown pellet was washed with 70% ethanol, dried at room temperature and was dissolved in TE (10 mMTrisCl, 1 mM EDTA, pH 8.0). DNA concentration was estimated spectrophotometrically. DNA was analyzed for restriction enzyme digestion analysis by Sau3A1 for 15 min.

References:

  • The AAPS Journal 2006; 8 (2) Article 28 (https://www.aapsj.org). Mol Biotechnol DOI 10.1007/s12033-007-0015-3
  • Solis, P. N., Wright, C. W., Anderson, M. M., Gupta, M. P. and Phillipson, J. D. 1993. A microwell cytotoxicity assay using Artemia salina (brine shrimp). Planta Medica, 59:250-252.
  • Carballo, J. L., Hernández-Inda, Z. L., Pérez, P. and García-Grávalos, M. D. 2002. A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products. BioMed Central Biotechnology, 2:17.
  • Barahona, M. and Sanchez-Fortun, S. 1999. Toxicity of carbamates to the brine shrimp Artemia salina and the effect of atropine, BW284c51, iso-OMPA, and 2-PAM on carbaryl toxicity. Environmental Pollution, 104:469-476.
  • Sajid, I., Yao, C. B. F. F., Shaaban, K. A., Hasnain, S. and Laatsch, H. 2009. Antifungal and antibacterial activities of indigenous Streptomyces isolates from saline farmlands: prescreening, ribotyping and metabolic diversity. World Journal of Microbiology and Biotechnology, 25:601-610.
  • Shirling, E. B. and Gottlieb, D. 1968. Cooperative description of type cultures of Streptomyces III. Additional species descriptions from first and second studies. International Journal of Systematic Bacteriology, 18:279-392.
  • Shirling, E. t. and Gottlieb, D. 1966. Method for characterization of Streptomyces species. Int. J. Syst. Citeseer.
  • Solis, P. N., Wright, C. W., Anderson, M. M., Gupta, M. P. and Phillipson, J. D. 1993. A microwell cytotoxicity assay using Artemia salina (brine shrimp). Planta Medica, 59:250-252.

 

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