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Ravi Kumar K. et al. J Sci Res Pharm, 2018;7(3):30-34

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Vol. 7, Issue 3, 2018 ISSN: 2277-9469

USA CODEN: JSRPCJ

Research Article

INSILICO ADME PROFILING OF CDK9 INHIBITORS

Ravi Kumar K. 1 _{*, Archana Giri} 1_{, Rama Rao Nadendla} 2

1 _{Centre for Biotechnology, Jawaharlal Nehru Technological University, Hyderabad - 500085, INDIA.} 2 _{Chalapathi Institute of Pharmaceutical Sciences Guntur - 522034, INDIA.}

Received on: 06-03-2018; Revised and Accepted on: 24-03-2018

ABSTRACT

S

everal drug targets have been identified in fighting against cancer. Inhibition of Cell cycle is one of the strategies used in anti-cancer research. CDKs [Cyclin Dependent Kinases] were found to be one of the promising drug targets. This work aims to find a potential molecule to inhibit CDKs that are involved in cell cycle progression. CDK 9 was chosen as potential drug target for cancer. Virtual screening was carried out against CDK 9 protein using Molecular Docking tools with molecules from ZINC database. Molecules were shortlisted based on their docking score, rerank score and energies. Insilico Toxicity and ADME [Absorption, Distribution, Metabolism and Excretion] analysis was carried to know the efficacy of the molecules before proceeding to invitro and invivo assays. Molecules under study were analyzed for ADME properties using Molinspiration, preAdmet and Swissadme servers. ADME profiles were evaluated and most of the molecules were found to be suitable for further studies. Insilico ADMET analysis is proved to be a good tool in drug discovery.

KEYWORDS: CDK, Cancer, Toxicity, ADME, Virtual Screening.

INTRODUCTION

M

ajority of deaths occurred in world is due to cancer. In search of developing strategies against cancer a huge amount of resources are being invested in various projects across the nations [1]_.

Drug designing is the important step in any drug discovery project. Insilico methods of drug design and development are proved to be efficient in saving lots of resources and time [2]_{. Cell cycle progression is}

one of the important steps involved in cell proliferation. Inhibition of Cell cycle progression using CDK as target is one of most suitable strategy to fight against cancer [3]_{. Over the last 30 years methods of}

computer aided drug design / discovery played a pivotal role in the development of therapeutic drugs [4]_{. The potential of any compound}

used in therapeutics depends not only on the physical and chemical properties but also on Pharmaco dynamics [PD] and pharmaco kinetics [PK] aspects of the compounds. Pharmacodynamics correlates health effect of drugs on an individual patient while pharmacokinetics records the course of Absorption, Distribution, Metabolism and Excretion of a given drug, both are interrelated.

Over the past 5 decades ADME played a major role in drug design process. ADME means absorption, distribution, metabolism and excretion which explain about the pharmacokinetics aspects of a drug molecule. There are several incidents reporting the attrition of drug discovery projects just because of the poor ADME profiles [5]_{. Therefore}

prior to synthesis and invivo studies, ADME profiling found to be more effective. Determination of ADME properties of compounds involves lot of experimental procedures to be followed which is time consuming and expensive. Therefore Insilco ADME models have been developed [6, 7]_{. In}

*Corresponding author:

Ravi Kumar. K

Centre for Biotechnology,

Jawaharlal Nehru Technological University, Hyderabad - 500085, INDIA.

* E-Mail: [email protected]

DOI: https://doi.org/10.5281/zenodo.1207094

the present study an insilio approach has been utilized in determining the ADME profiles of the compounds known to be CDK 9 inhibitors.

MATERIALS AND METHODS

Insilico ADME analysis:

The Compounds used in this work were found to be inhibiting CDK 9 [8] _{protein as studied earlier using virtual screening studies} [9]_.

The molecules were short listed based on their docking scores, ranking scores and rerank scores [9]_{. Virtual screening was carried with}

molecules available in ZINC database [10]_.

Experimental:

The test compounds to be used as potential CDK 9 inhibitors used in the study are listed in Table 1 and their structures are shown in Figure 1.

Calculation of ADME properties:

All the 2D molecular structures were drawn online at Molinspiration server [11]_{. ADME properties of test set compounds were}

calculated using Molinspiration [11]_{, PreAdmet} [12] _{and Swiss ADME tools} [13]_.

1. Plasma Protein binding:

A drug is more efficient if it is free to traverse across membranes and reach the target rather binding with plasma proteins. Compounds with more than 90% PPB values are more prone to plasma protein binding thereby less effective and vice versa. %PPB value less than 90% is more effective.

2. Blood brain barrier:

3. Human intestinal absorption:

One of the important parameter in ADME study is drug's intestinal absorption. When a drug is administered orally it has to be absorbed by intestine for further metabolism of the drugs. Human intestinal absorption was analyzed by combining the bioavailability and absorption generated from the ratio of excretion or excretion in urine, bile and feces together [15]_.

4. Skin Permeability:

In drug discovery the skin permeability of the compound is an important determinant factor with reference to adverse drug response in case drugs taken orally to determine in case of accidental contact with skin and the skin permeability in case of the drugs to be taken transdermally where the skin penetration is an important aspect. Preadmet asses the skin permeability of a compound the result value is given as logKp. Kp [cm/hour] is defined as: Kp= Km*D/h where Km is

distribution coefficient between stratum corneum and vehicle, and D is average diffusion coefficient [cm2/h], and h is thickness of skin [cm] [16]_.

5. Caco2 cell permeability:

For assessing the oral absorption Caco 2 cell line and MDCK cell line models have been used [17]_{. Caco-2 cells are derived from human}

colon adenocarcinoma and possess multiple drug transport pathways through the intestinal epithelium.

6. MDCK cell permeability:

MDCK cell refers to Madin-Darby canine kidney cell. Compared to Caco -2 cells the life span of MDCK cells life span is less, there seems to be highly correlated. Hence along with Caco 2 cell lines MDCK cell system is also being used as a tool for rapid permeability screening. MDK cell permeability values < 25 is considered as low permeable, 25-500 - medium permeable and values >500 are considered as molecules with high permeability.

Table No. 1: Test Compounds used in study

S. No. Name

i N-[[3S]-3-[4-fluorophenyl]-3-[2-furyl]propyl]-3-[5-methyl-2-furyl]-1H-pyrazole-5-carboxamide

ii N-[[3R]-3-[4-fluorophenyl]-3-[2-furyl]propyl]-3-[5-methyl-2-furyl]-1H-pyrazole-5-carboxamide

iii N-[4-methyl-3-[[3-[4-nitrophenyl]-1,2,4-oxadiazol-5-yl]methylamino]phenyl]cyclopropanecarboxamide

iv 2-[2-[3-amino-4-methoxy-phenyl]imidazo[1,2-a]pyridin-8-yl]oxy-N-[2-furylmethyl]acetamide

v N-[3-[[7-methoxy-2-oxo-chromen-4-yl]methylamino]-4-methyl-phenyl]cyclopropanecarboxamide

vi 2-[3-[3,4,5-trimethoxyphenyl]-1H-pyrazol-4-yl]-1H-benzimidazole-5-carboxylic

vii 3-[5-[[Z]-[[5-amino-4-cyano-3-methyl-thiophene-2-carbonyl]hydrazono]methyl]-2-furyl]benzoic

viii N'-[4-nitrophenyl]-N-[3-[2-phenylimidazol-1-yl]propyl]oxamide

ix [4R]-3-[2-hydroxy-5-methyl-phenyl]-4-[3-hydroxyphenyl]-5-[3-methoxypropyl]-1,4-dihydropyrrolo[4,3-d]

x

[i] [ii] [iii]

[iv] [v] [vi]

[x]

Fig. 1: Structures of Test set Compounds

Other Parameters studied like MiLogP or WLogP, TPSA, natoms, MW, nON, nOHNH, nviolations, nrotb, and volume as per Molinspirations server. Using Swissadme server egg plot depiction [18] was made using TPSA vs WLogP values. It indicates the Blood brain barrier [BBB] penetration and Intestinal Absorption capacities [Table 3 & figure 2].

RESULTS & DISCUSSION

F

or the test compounds the calculated properties as determined by Molinspiration and Preadmet Servers were tabulated in Table 2 and 3.

Table No. 2: Compound Properties analyzed by Molinspiration server

Property Parameter

Compound

1 2 3 4 5 6 7 8 9 10

miLogP 3.67 3.67 3.69 2.21 3.69 3.05 3.73 2.25 2.92 2.79

TPSA 84.06 84.06 125.87 104.04 125.87 122.37 141.72 121.85 98.68 87.69

natoms 29 29 29 29 29 29 28 29 29 29

MW 393.42 393.42 393.4 392.42 393.4 394.39 394.41 393.4 393.44 393.44

nON 6 6 9 8 9 9 8 9 7 7

nOHNH 2 2 2 3 2 3 4 2 3 2

nviolations 0 0 0 0 0 0 0 0 0 0

Nrotb 7 7 7 7 7 6 5 8 6 7

volume 345.54 345.54 340.19 344.44 340.19 336.15 325.5 344.58 353.99 354.96

Table No. 3: ADME parameters analyzed by preAdmet Server

Parameter Compound

1 2 3 4 5 6 7 8 9 10

BBB 1.81 1.81 0.06 0.03 0.06 0.07 0.11 0.05 0.68 0.57

Caco2 17.07 17.07 20.04 17.70 20.04 49.06 0.39 20.42 8.55 20.41

HIA 90.97 90.97 90.80 95.74 90.80 99.14 78.27 93.03 86.73 90.62

MDCK 0.35 0.35 1.57 14.07 1.57 2.01 0.18 1.80 5.58 4.95

PPB* 88.44 88.44 89.70 77.97 89.70 61.92 87.50 94.83 86.46 84.83

SP* -3.91 -3.91 -3.55 -4.01 -3.55 -4.08 -3.28 -3.37 -4.25 -4.15

*Plasma Protein Binding [PPB], Skin Permeability [SP]

1. Plasma Protein Binding: Preadmet analysis of the test set have been found almost all the molecules are having %PPB less than 90% except 8th molecule [table3].

2. Blood brain barrier: 0.04 - 1.81. [table 3] Almost all the molecules are having values less than 1 which means low absorption to CNS and only two molecules 1 and 2 are having values around 1.81 which is still acceptable as those compounds are intermediary in absorption to CNS. As the present work is on cancer and there is no CNS penetration of compounds is needed. Hence these compounds can be considered with reference to BBB penetration.

3. Human intestinal absorption: Preadmet asses the intestinal absorption capacity of the tested compounds and values ranging 0-20 % found to be poorly absorbed and 20-70 % moderately absorbed, values ranging 70-100 % are found to be compounds with greater absorption. Compounds 7 and 9 are having 78.2 and 86.7 %HIA and rest all are 90% and above % HIA values. Hence almost all molecules are found to be efficiently absorbed by human intestine.

4. Skin Permeability: all the compounds found [table 3] to be poorly permeable to skin as all compounds have Kp negative values. As the compounds are to be taken orally their skin permeability is to be low and accidental contact will not be having any effect on the skin.

5. Caco2 cell permeability: Values less than 4 are poorly permeable, 4-70 are moderately permeable and more than 4-70 are considered as highly permeable. In the present study preadmet has determined compound 7 is poorly permeable and rest all compounds are in the range of 17 - 50 which means almost all compounds are moderately permeable to Caco 2 cell lines.

Fig. 2: Egg plot denotes bioavailable region of property space with respect to wlog P and TPSA

[Yellow - BBB, White - Intestinal absorption, Grey - Poor intestinal absorption]

As discussed earlier [19] _{Caco2 cell line data alone cannot be}

considered in evaluating gastro intestinal absorptions. In the present work the Caco 2 cell line absorption is showing poor where as human intestinal absorption capacity of all the molecules shown very high. Hence more studies have to be carried out before considering the compounds.

Lipophilicty is measured as a compound’s distribution across octanol vs water phase and is expressed as log of concentration rations of those phases [partition coefficient]. A logP value not more than 5 is highly desirable. This is also a part of Lipinski rule of five. All the test compounds have shown logP values under 5.

TPSA [Topological polar surface area [TPSA] can be defined as the total area in space covered by polar atoms like hydrogen atoms, oxygen atoms and nitrogen atoms present in the chemical structure. It is a measure of drub's ability in cell permeation.

Other parameters like natoms [No of Atoms], MW Molecular Weight, nON, nOHNH, nviolations, nrotb [no of rotatable bonds] and volume as analyzed by Molinspiration server are found to be in good terms.

Swissadme server used comparative information derived from poorly and highly absorbed drugs to model human passive intestinal absorption. Calculated passive intestinal absorption of drugs was displayed as a bi-plot [Boiled Egg Plot or Egg plot]. Bi-plot utilizes information generated from Wlog P [Y-axis] and Total Polar Surface Area [TPSA] [X-axis] mentioning well-absorbed molecules in space [fig.5]

Inside circle [yellow] depicts BBB- blood brain barrier, none of the compounds are in this region. The white region which is outer to yellow depicts the human intestinal absorption. Almost all compounds lies in this white area. Only molecule 7 is lying outside [grey area] which indicates poor intestinal absorption. From ADME profiling it can be concluded that Molecule 7 will be poorly absorbed even though it is in top 10 compounds of virtual screening studies. This is the advantage of Insilico adme profiling. The present graphical representation provides better understanding about absorption profile of novel molecules

CONCLUSION

I

t was reported [20] _{that generally it takes around 8 years in}

project towards the success. The present study that overall Insilico ADME modeling found to be a good alternative over In vitro / In vivo models of ADME. Almost all the compounds were found to be suitable as potential candidate molecules as CDK 9 inhibitors. Once a good set of compounds were screened based on ADME modeling further In vitro and In vivo studies can move the discovery process further.

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How to cite this article:

Ravi Kumar K. et al. INSILICO ADME PROFILING OF CDK9 INHIBITORS. J Sci Res Pharm 2018;7(3):30-34. DOI:

https://doi.org/10.5281/zenodo.1207094

Conflict of interest: The authors have declared that no conflict of interest exists.