ACTIVITY 09

In Silico Analysis of Cytotoxic Nucleoside Analogues

Liwas, J.K.1, Dela Cruz, M.F. 1, Likigan, A.G. 1, Marcos, D. 1, Mercado, J.B. 1, & Reyes, K. 1

1Student, Department of Medical Laboratory Science, School of Natural Sciences, Saint Louis University

In Partial Fulfillment of the Requirements for the Subject

Biochemistry for Medical Laboratory Science
MLS 211L

November 28, 2019

 INTRODUCTION
Nucleosides are structural subunit of nucleic acids. They are glycosylamines that form nucleotides
which are the building blocks of nucleic acids. Structurally, they consist of a molecule of sugar linked to a
nitrogen-containing organic ring compound. In the most important nucleosides, the sugar is
either ribose or deoxyribose, and the nitrogen-containing compound is either a pyrimidine that is a cytosine,
thymine or uracil or a purine that is an adenine or a guanine (Nucleoside Biochemistry, 2019).
Nucleosides and nucleoside analogues are used in antiviral and anticancer drug therapy representing the
largest class of small molecule-based antivirals. These drugs generally are hydrophilic in nature and require
specialized transport proteins to facilitate their uptake and/or release from the cell. Modified nucleosides are
important molecules which display antiviral and antitumoral activity due to their capacity for inhibiting DNA
synthesis (Taft, 2009).
This experiment is focused in the in silico analysis of cytotoxic nucleoside analogues. The
experiment aimed identify the molecular descriptors of nucleoside analogues which are which are related
to their cytotoxic activities and to construct an approximated multiple linear regression model of the
structure-activity relationship of nucleoside analogues and their cytotoxic activities.

FLOWCHART OF PROCEDURES WITH INFOGRAPHICS

The flowchart below presents the procedures taken for the completion of the in-silico analysis of
nucleoside analogues.

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 RESULTS AND FINDINGS
This chapter presents all findings during the performance of in-silico analysis through collated
tables and figures.

Nucleoside Analogue Canonical SMILES

1 Nc1ncnc2n(CC3C[C@@H](O)C4C=CC=CC34)cnc12
2 OC[C@@H]1C[C@@H](Cn2ccc(=O)[nH]c2=O)c3ccccc13
3 Nc1ccn(C[C@@H]2C[C@@H](CO)c3ccccc23)c(=O)n1
4 O[C@@H]1CC(Cn2cnc3c4NCC[CH3]n4cnc23)C5C=CC=CC15
5 CC(C)Nc1ncnc2n(CC3C[C@@H](O)c4ccccc34)cnc12
6 O[C@@H]1CC(Cn2cnc3c(O)ncnc23)c4ccccc14
7 COc1ncnc2n(CC3C[C@@H](O)c4ccccc34)cnc12
8 CCOc1ncnc2n(CC3C[C@@H](O)c4ccccc34)cnc12
9 O[C@@H]1CC(Cn2cnc3c2ncnc3c4ccccc4)c5ccccc15
10 CC(C)Oc1ncnc2n(CC3C[C@@H](O)c4ccccc34)cnc12
Table 1. Canonical SMILES of Nucleoside Analogues
Table 1 presents the Canonical Simplified Molecular Input Line Entry System (SMILES) of each
nucleoside analogues after being drawn in MolView. These SMILES were then plugged in to ChemDes for
the retrieval of their respective chosen molecular descriptors and charges.

Data from ChemDes

Nucleoside Activity
(log IC50-1) Molecular Descriptors Charges
Analogue LDI QHmax QCmax QOmin QOmax
TPSA Hy MR
1 -2.53 89.85 -3.137 79.109 0.199 0.210 0.165 -0.392 -0.392
2 -2.59 75.09 -3.241 75.382 0.204 0.210 0.328 -0.396 -0.248
3 -2.87 81.14 -3.439 76.530 0.187 0.210 0.349 -0.396 -0.246
4 -2.32 62.80 -3.439 92.775 0.175 0.210 0.080 -0.392 -0.392
5 -2.20 75.86 -3.439 92.842 0.196 0.211 0.165 -0.388 -0.388
6 -2.42 84.06 -3.137 75.748 0.212 0.295 0.242 -0.492 -0.388
7 -2.54 73.06 -3.243 80.365 0.226 0.211 0.245 -0.479 -0.388
8 -2.33 73.06 -3.744 85.252 0.222 0.211 0.245 -0.476 -0.388
9 -1.45 63.83 -3.797 99.519 0.149 0.211 0.163 -0.388 -0.388
10 -2.40 73.06 -3.439 89.847 0.149 0.211 0.245 -0.473 -0.388
Correlation -0.5884 -0.8669 0.8218 -0.6024 -0.0423 -0.5662 0.1825 -0.5144
Table 2. Molecular Descriptors, Charges, Activity, & Correlation Analysis of Nucleoside Analogue
Table 2 presents the anticancer activity of each nucleoside analogue in terms of its logarithmic
1
value of the reciprocal of their half maximal inhibitory concentration ( ). Therefore, the closer the activity
IC50
of the nucleoside analogue to 0, the more potent it is – leading to a better anticancer activity of the analogue.
Hence, nucleoside analogue 9 had the best anticancer activity of the 10 nucleoside analogues, and
nucleoside analogue 3 was the least – being the analogue that needed the highest concentration of the 10
analogues just to inhibit half of the cancer activities. The succeeding columns after the activity present the
molecular descriptors (3) and charges (5) of the nucleoside analogues after their respective canonical
SMILES were plugged in to ChemDes.
The same table also present the correlation of analysis between the activity with the molecular
descriptors and charges. Evaluating their absolute correlation values, the hydrophilic index (Hy) had the
highest correlation with a substantial correlation; followed by the molar refractivity (MR) having also a
substantial correlation and a direct relationship with the activity; with the local dipole index (LDI) having the
3rd highest value with a moderate correlation. Between these 3 data, Hy and LDI have an inverse
relationship with the activity of the nucleoside analogues – as one increase, the other decreases; and only
MR has a direct relationship with the values of the activity of the nucleoside analogues.

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 Table 3. Coefficient Values for the Formulation of a Regression Model

Table 3 presents the coefficient values used for the formulation of a multiple regression model
based on the 3 molecular descriptors and charge that had the highest correlation values. Being significant
at 0.02 (< α = 0.05), the uncorrected regression model y = -1.24(Hy) + 0.01(MR) – 1.01(LDI) – 6.96 takes
into account only 63.63% of determining the nucleoside analogue activities (y variable) given the Hy, MR,
and LDI (x variables). For every 1 unit increase of Hy, the activity of the nucleoside analogue decreases by
-1.24 units; for every 1 unit increase of MR, the nucleoside analogue activity increase by 0.01 unit; for every
1 unit increase of LDI, the activity decreases by 1.01 units; and when all 3 variables are 0, the activity of
the nucleoside analogue is at a constant of -6.96 units. Observing the p-values of each variable, it is seen
the p-values of the x variables are greater than 0.05. Hence, the corrected regression model would lead to
y = -6.96 only.

DISCUSSION OF RESULTS
This chapter presents all interpretations of the findings after the performance of in silico analysis
through related literatures.
Nucleoside analogues characterized by cytotoxicity are clinically important anticancer drugs. The
mode of action exhibited by nucleoside analogues are similar to that of antimetabolites. Nucleoside
analogues may also compete with physiologic nucleosides and interact with other intracellular targets to
promote cytotoxicity. Both purine and pyrimidine analogues are used as cytotoxic agents to treat tumors.
The use of drugs derived from nucleoside analogues, however, is limited due to drug resistance therefore
raising the demand for development of new antimetabolites that would work as efficient as or more effective
than the current treatments (Galmarini, Popowycz, & Joseph, 2008). Furthermore, a demand on new ways
of administration of nucleoside analogues to cancer patients necessitates scientists today to study
nucleoside analogues. In this regard certain molecular descriptors are observed via in silico analysis and
Quantity Structure-Activity Relationship to determine which descriptors could actually account for the
cytotoxicity and anti-cancer properties of the given nucleoside analogues. By being able to determine the
relationship of the molecule’s structure to its anti-cancer property, an analogues cytotoxic action in vitro and
in vivo can be predicted.

In this experiment, MolView was used to obtain the canonical SMILES of the six nucleoside
analogues. The canonical SMILES of the six nucleoside analogues were then plugged in to ChemDes and
the molecular descriptors were obtained, namely: topological polarity surface area (TPSA), hydrophilic
index (Hy). The charge of these nucleoside analogue was also obtained, namely: local dipole index (LDI),
most positive charge on hydrogen atom (QHMax), most positive charge on carbon atom (QCMax), most
negative charge on oxygen atom (QOmin), and the most positive charge on oxygen atom (QOmin).
Correlation is a statistical tool used to show the relationship of 2 variables – what type of relationship
they have and the strength of correlation. In this experiment, the IC50 values of the 6 nucleoside analogues
were subjected to correlation analysis with their molecular descriptors with the use of Microsoft Excel.
Hydrophilicity index (Hy), Molar Refractivity (MR), and Local Dipole Index (LDI) are the molecular
descriptors that had a substantial correlation with the IC50 of the metabolites with, a value Hy = -0.87, MR
= 0.82 and the LDI = -0.60.

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 The Hydrophilicity Index (Hy) is an empirical index which allows the measurement of the
hydrophilicity of a compound (Tedeschini & Consonni, E-H, 2008). This molecular descriptor had the higest
correlation with cytotoxic activity. According to Singh, Singh, Kundu, Bansal, & Bajaj (2013), the high
hydrophobicity causes many disrupt the membranes of cells, leading to better cytotoxic activities. Also,
according to Sıdır and Sıdır (2013), the hydrophobic parameter is mainly responsible for cytotoxicity. In
other words, the more hydrophobic a nucleoside is, the higher the cytotoxic activity is.
The Molar Refractivity (MR), on the other hand, provides information about the molecular volume
and polarizability of a liquid (Tedeschini & Consonni, I-Q, 2008). According to study done by Dimmock et
al. (1998), the molecular refractivity is inversely proportional to the cytotoxic activity. Finally, the Local
Dipole Index (LDI) provides the average of the charge differences over all the bonded pairs (Tedeschini &
Consonni, I-Q, 2008).
Regression analysis is also a statistical tool that helps one predict Y variable given X variables
through the formulation of a regression model. With the Hy, MR and LDI values represented by X and IC50
value represented by Y, the formulated uncorrected regression model is y= -6.96 – 1.24 (Hy) + 0.01 (MR)
– 1.01 (LDI), which is not statistically significant (p-value of Hy = 0.30 > 0.05; p-value of MR = 0.74 > 0.05;
p-value of LDI = 0.82), correctly predicts the IC50 only 64.63% of the time given Hy, MR and LDI.
Furthermore, for every 1 unit increase of Hy, IC50 decreases by 1. 24 µL, foe every 1 unit increase of MR,
IC50 decreases by 0.01 µL, and for every 1 unit increase of LDI, IC50 decreases by 1.01 µL. When the Hy,
MR, and LDI is 0, IC50 is at a constant of -6.96 µL.
CONCLUSION
The structure-activity relationships of nucleoside analogues with antiviral activity were studied
utilizing structure-activity maps. Structure activity maps are chemical structures quantified by molecular
descriptors, plotted against their biological activities. The molecular descriptor number of atoms and bonds
of a molecule was used to quantify the chemical structures.
Structure activity maps were used systematically to identify the effects of chemical modification on
the antiviral activity of nucleoside analogues and to determine the site and type of modifications for
improved activity and reduced toxicity of potential antiviral agents. Molecular descriptors to quantify the
chemical structure is the integer value of number of atoms and bonds of molecule that denotes the number
of nonhydrogen atoms and bonds o molecule as stated by Parakulam, lesniewski, Marquis, and Tsai (1987)
For the construction of multiple linear regression model, the 3 chosen molecular descriptors with
highest correlation was Hydrophilic Index (Hy), Molar Refractivity (MR) and Local Dipole Index (LDI) which
was further utilized in constructing linear regression.
y= - 6.96 - 1.24(Hy) + 0.01(MR) - 1.01(LDI) The regression model formula would mean that: For
every one unit increase of Hy, there is a 1.23611 decrease in inhibition; for every one unit increase of MR
there is a 0.007836 increase in inhibition and lastly for every one unit increase in LDI there is 1.00824
decrease in inhibition.

RESEARCH QUESTION/S
What is the role and application of the concept of aromaticity to the properties of nucleosides and
nucleotides?
According to Chaudhuri, Ren and Kool (1997), synthesis, structure and DNA incorporation of
a class a novel aromatic C-deoxynucleosides in which benzenes and larger polycyclic aromatics serve
as DNA base analogues.
Weinfeld, Soderlind, and Buchko (1992) stated that stacking between aromatic amino acids
and nucleic acid bases may play an important role in the interaction of enzymes with nucleic acid
substrates.

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 They have examined the requirement for DNA base aromaticity of five enzymes that act on a
single-stranded DNA, t4 polynucleotide kinase, nucleases P1 and S1, and snake venom and calf spleen
phosphodiesterase by comparing their kinetics of reaction with a series of dinucleoside
monophosphates containing thymidine or a ring-saturated derivative. The modified substrate contained
either cis-5R, 6S-di-hydro-5, 6-dihydroxythymidine (thymidine glycol) or a mixture of the 5R and 5S
isomers of 5,6-dihydothymidine. It was observed that for all enzymes, except snake venom
phosphodiesterase, the parent molecules were better substrate than dihyrothymidine derivatives, while
the thymidine glycol compounds were significantly poorer substrates.snake venom phosphodiesterase
acted on the unmodified and dihydrothymidine molecules at almost sane rate. These results imply that
for all the remaining enzymes base aromaticity I a factor in enzyme-substrate interaction.
Protein recognition of DNA involves a combination of electrostatic, Van Der Waals,
hydrophobic and hydrogen-bonding interactions. Several studies have shown that the presence of
aromatic amino acids in both small and large peptide chains enhance binding to single stranded DNA
by interaction with nucleic acid bases in the form of either stacking or other hydrophobic interactions.
Clearly, where stacking is involved, aromaticity of the bases must play an important role.

REFERENCES
Chaudhuri, N., Ren, X., & Kool, E. (1997). C-Nucleosides Derived from Simple Aromatic Hydrocarbons.
Synlett, 4, 341-347. doi: 10.1055/s-1997-788

Dimmock, J. R., Kandepu, N. M., Hetherington, M., Quail, J. W., Pugazhenthi, U., Sudom, A. M., &
Balzarini, J. (1998). Cytotoxic Activities of Mannich Bases of Chalcones and Related Compounds.
Journal of Medicinal Chemistry, 41 (7) 1014-1026. doi: 10.1021/jm970432t
Galmarini, C., Popowycz, F., & Joseph, B. (2008). Cytotoxic Nucleoside Analogues: Different Strategies
to Improve their Clinical Efficacy. Current Medicinal Chemistry, 15(11), 1072–1082. doi:
10.2174/092986708784221449
Nucleoside Biochemistry (2019). Retrieved from Encyclopedia Britannica:
https://www.britannica.com/science/nucleoside

Parakulam, R., Lesniewski, M., Marquis II, M., & Tsai, C. (1999). Structure-Cytotoxicity/Antiviral Activity
Relationship Studies of nucleoside analogs using structure - activity maps. AAAI Technical
Report SS-99-01.

Sıdır, Y., & Sıdır, İ. (2013). Quantitative structure activity relationships of cytotoxicity effect on various.
Bitlis Eren Univ J Sci & Technol, 3, 9-14. ISSN: 2146-7706
Singh, M., Singh, A., Kundu, S., Bansal, S., & Bajaj, A. (2013). Deciphering the role of charge, hydration,
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amphiphiles. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1828 (8), 1926-1937. doi:
10.1016/j.bbamem.2013.04.003
Taft, D. (2009). Drug Excretion. Retrieved from ScienceDirect: https://doi.org/10.1016/B978-0-12-369521-
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Tedeschini, R., & Consonni, V. (2008). Handbook of molecular descriptors. WILEY‐VCH Verlag GmbH.
Online ISBN: 9783527613106
Weinfeld, M., Soderlind, K., & Buchko, G. (1993). Influence of nucleic acid base aromaticity on substrate
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