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In Silico Drug Design of Biofilm Inhibitors of Staphylococcus epidermidis

by Al-mulla, Aymen Faraoun, MS


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B

Figure 4.39 Effect of gradient concentrations of Ibuprofen on: A-bacterial growth; B-
slime production

G- Thymol:
Thymol (also known as 2-isopropyl-5-methylphenol) is a
natural derivative of cymene found in thyme oil and extracted from Thymus
vulgaris (common thyme) and various other kinds of plants as a
white crystalline substance of a pleasant aromatic odor and
strong antiseptic properties. It was selected as an antibiofilm as its
hypothetical binding energy to the sarA protein was (-5.4 kcal/mol).
Figures (4.40 A,B) show that bacterial growth and biofilm production
increased after the addition of Thymol, which may indicate that the bacteria
can consume it as a source of nutrient. The correlation coefficients for VC
and slime OD with concentration were -0.23, 0.66 respectively.


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Log
cfu/ml
9
8
7
6
5
4
3
2
1
0

0 100 200 300 400 500 600 700
Concentration (µg/ml)

A

B

Figure 4.40 Effect of gradient concentrations of Thymol on: A-bacterial growth; B-
slime production


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In recent years mounting problems related to antibiotic-resistant bacteria
have resulted in the prediction that we are entering the post-antibiotic era.
A way of preventing such a development would be to introduce novel
antibacterial medicines with modes of action distinct from conventional
antibiotics. Recent studies of bacterial virulence factors and toxins have
resulted in increased understanding of the way in which pathogenic
bacteria manipulate host cellular processes.
This knowledge may now be used to develop novel antibacterial
medicines that disarm pathogenic bacteria. The industrial failure to meet
the public health needs of new antibacterial drugs indicates that novel
approaches are needed in drug discovery. Traditional antibiotics kill or
inhibit the growth of bacteria; they are either bactericidal or bacteriostatic.
They have an important drawback as they not only attack bacteria causing
the infection but also other bacteria. Intestinal, airway and skin bacteria
build up a normal flora that is essential for our well-being when kept in
balance. If the normal flora is disturbed by antibiotics, resistant bacteria are
allowed to grow.
The arguments are that resistance to compounds targeting the virulence
factors cannot evolve and spread in the resident flora, as these bacteria lack
virulence targets. It is also proposed that resistance to virulence blocking
agents is likely to result in nonfunctional virulence systems, and thus
nonvirulent bacteria. Further, as long as the target remains extracellular,
resistance cannot emerge through the activity of bacterial efflux pumps.
Finally, there will be a low selective pressure for mutations affecting the
specific interaction between the drug and a virulence factor, as virulence
blockers should have low effect on bacterial growth. (Keyser et al., 2008).
The experiments were carried out not just to estimate biofilm inhibition,
but they were combined with estimation of VC. It was forwarded to use


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compounds that inhibit or disturb the function of sarA without affecting
bacterial cell growth. This point seems quite important, as it leaves no
opportunity of resistant sub-clones to dominate the population, which is the
main feature of antibiotic treatment. In the latter case, a certain portion of
population is resistant but is rare or at a very low level, so using compounds
killing the normal (predominant) cells would offer the opportunity for such
sub-population to dominate. This represents the story behind the
development of resistant strains (Keyser et al., 2008).
The results above may lead to a conclusion that Acetaminophen and
Acetylsalicylic Acid gave 100% antibiofilm activity, and that Ibuprofen
and Acetic Acid gave 35%, 25% antibiofilm activity respectively. The
other molecules were poor biofilm supressors.
This leads to proposal that Non Steroidal Anti Inflammatory Drugs
(NSAID) constitute a good family for searching antibiofilm drugs. Farber
and Wolff (1992) reported that Salicylic Acid inhibited adherence (55%)
and biofilm production of S. epidermidis (Farber and Wolff, 1992). A
second study by Farber et al. (1995) further illustrated that NSAIDs,
including sodium salicylate, inhibit S. epidermidis and P. aeruginosa
biofilm production on contact lenses, lens cases, and commonly used
medical polymers in a dose-related manner. This manner has also been
observed by other authors (Tomlinson et al., 2000; Bandara et al., 2004).
Alem and Douglas (2004) found in their studies on Candida albicans that
seven of nine (NSAID) drugs tested at a concentration of 1 mM inhibited
biofilm formation. Aspirin, etodolac, and diclofenac produced the greatest
effects, with aspirin causing up to 95% inhibition. Celecoxib,
nimesulide, ibuprofen, and meloxicam also inhibited biofilm formation,
but to a lesser extent. Aspirin was active against growing and fully mature


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(48-h) biofilms; its effect was dose dependent, and it exhibited significant
inhibition (20 to 80%) at pharmacological concentrations.
Abd El-Aziz et al. (2012) found that Salicylate at a concentration of 10
μg/ml reduced biofilm synthesis by 57.01% and eradicated pre-adhered
biofilms by 29.19% while at 100 μg/ml biofilm synthesis was reduced by
68.35% and pre-formed biofilms was disrupted by up to 62.73%.
In comparison with the previous study, the results of Acetylsalicylic Acid
at therapeutic levels (50-200 μg/ml) involved inhibition to pre-formed
biofilm by 22%. This difference in results is due to difference in
experiment conditions, in bacterial genera (in the previous study P.
aeruginosa was used) and in the intensity of bacterial biofilms as we have
chosen the most potent biofilm producer from nine isolates.
An important point must be discussed here. In vitro studies and results
must not be taken as final results for clinical applications because:
1. Difference in environmental conditions may affect the results, where lab
tools, nutrient media, temperature and solubility status differ from plasma
conditions, body circulation, body temperature, body defenses.
2. Bacterial behavior in lab differs from that in human body.
3. Drug molecule characteristics in lab differ from those in human body
where they may undergo degradation, conformational changes, rearrangement,
plasma protein binding.
Therefore, all lab experiments are considered as a beginning signal and
must be completed with in vivo studies.
This study did not only propose new drug line as antibiofilm, but also
adopted a new strategy for drug design and identified a new target to be hit
by drug designers to suppress biofilm production .


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In this study four of seven molecules were proposed to suppress biofilm
production by inhibiting sarA protein activity. This means that the percent
of success in drug design was about 60%. This percent is good if we put in
mind the hypothetical processes and prediction and modeling of protein
and the error percent in each drug design step.
Finally, this is an in Silico and in vitro study, where the concentrations
of the molecules as antibiofilms do not meet the pharmacological limits for
human use (maximum therapeutic plasma concentrations for
Acetaminophen, Acetylsalicylic Acid and Ibuprofen are 150, 225, 50
µg/ml respectively) (Mueller and Lieberman, 1970; Hall et al., 1988;
Rumack, 2002). However, it gives an initial view of novel antibiofilm
drugs (where biofilm has been used mainly as a bioassay model for drug
design) and can be completed with in vivo studies in future. On the other
hand, there are no limitations on using these compounds in other aspects
away from human body, as an antiseptic or medical equipment surface
protectant.


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Conclusions
and
Recommendations


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Conclusions and Recommendations
Conclusions:
1. Structure based drug design strategy using Ligand Based Virtual
Screening had a success score about 60% .
2. Acetaminophen, Acetylsalicylic Acid, Ibuprofen and Acetic Acid can be
used as external antibiofilm molecules for surfaces and medical
equipments in 11 mg/ml, 1.6 mg/ml, 0.6 mg/ml and 1 mg/ml
concentrations respectively.
3. Non-Steroidal Anti-Inflammatory Drugs family can be offered as a
useful library for antibiofilm future investigations.
4. The sarA protein can be used as a good target for biofilm suppression.
5. The Congo Red Agar method can be excluded from biofilm estimation
assays.
6. The generation times of isolates of S. epidermidis were estimated to be
48-71 min.


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Recommendations:
1. Increasing and expanding in Silico drug design in the drug industry in
Iraq to lessen the stress of big companies.
2. The sarA protein can be used as a good target for biofilm suppression
and bioassay.
3. Further in vivo studies for Acetaminophen, Acetylsalicylic Acid,
Ibuprofen and Acetic Acid to estimate the best therapeutic dose as internal
antibiofilm agents may be carried out.
4. NSAID family can be used as a library for screening antibiofilm agents.


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ةصلاخلا
ةيودلأا داجيلإ ةيرورضلا ةثيدحلا تاهجوتلا دحا يبوساحلا ةيودلأا ميمصت لثمي
هذه يف بوساحلا ةطساوب يئاودلا ميمصتلا تايجيتارتسا تمدختسا.اهريوطتو
Staphylococcus ايرتكب يف ةيويحلا ةيشغلأل تاداضم ىلع لوصحلل ةساردلا
هذه يف ربكلأا ةوارضلا لماع ةيويحلا ةيشغلأا جاتنا دعي ثيح epidermidis
.ايرتكبلا
زيفحت يف دعاسي هنوكل يئاودلا ميمصتلا جمارب يف فدهك )sarA( نيتورب ريتخا
يأ ،ايرتكبلا يف ةيويحلا ةيشغلأا نيوكت نع لولأا لوؤسملا )icaADBC( نوريبوا

S.
.ةيويحلا ةيشغلأا نيوكت ةرهاظل ةيساسلأا تامظنملا دحا نوكي
ايرتكبب ةباصلإا صيخشتل نييقارع ىضرم نم ةنيع
رهجملاب تصحف ثيح 2182/2/21 ىلا 2182/81/81
ةلئاعل يمتنت اهنم ةلزع 18

69

ىلع لوصحلا مت
نم ةدملل epidermidis
نا ةجيتنلا تناكف مارغ ةغبصب اهغيبصت دعب يئوضلا
ةبسن تناك ةيويحوميكلا صوحفلاب تلازعلا هذه صحف دعبو .ةيدوقنعلا تاروكملا
ىلع لوصحلا مت ةيليمكت صوحف ءارجإب مث ،%87 اهنم S.epidermidis ايرتكب
.ةيويحلا ةيشغلأل ةجتنم ايرتكبلا نم تلازع
ةئيزجلل داعبلاا يثلاث ميمصتلاب لمعلا أدب يئاودلا ميمصتلل بوساحلا جمارب مادختسابو
ىلع لوصحلاو نيتوربلا ةجذمنل(RaptorX) عقوم مادختساب كلذو )sarA( فدهلا

جمانرب مادختساب )Pharmacophores(
6

.عقاولل برقلأا جذومنلا
ةيضارتفلاا تائيزجلا ميمصت ىرج
مث نمو )Discovery studio) جمانرب ةمزح نمض )Hip Hop generator(
)Virtual screening( تائيزجلل يضارتفلاا حسملا ةيلمع يف اهمادختسا
اهل ةيوايميك ةئيزج 888
ةيلباقلا اهل ةيوايميك ةئيزج
ةجيتنلا تناك ثيح )ZincPharmer( عقوم مادختساب
28 كانه تناكو .ةيضارتفلاا تائيزجلل ةبيرق تافصاوم
)EADock( ثحبلا كرحم لامعتسا نم ترهظ )sarA( نيتوربب طابترلاا ىلع

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