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Please complete and remit to Editorial office, Online Journal of Bioinformatics

Author(s): Satish kumar(Msc), Jyoti Pathak(Msc), Asha(Msc), Rajib Bandopadhyay(PhD)

ID Number: QA218

Title: Farnesyl pyrophosphate inhibition by drug binding to the allosteric site : An approach to Progeria therapy

The Editorial board must ensure that the OJB publishes only papers which are scientifically sound. To achieve this objective, the referees are requested to assist the Editors by making an assessment of a paper submitted for publication by:

(a) Writing a report as described in GENERAL STATEMENTS (Below),
(b} Check the boxes shown below under 1. and 2.

( YES or NO) [N.B.A "NO" assessment must be supported by specific comment in the report.
(c) Make a recommendation under 3.

The Editor-in-Chief would appreciate hearing from any referee who feels that he/she will be unable to review a manuscript within four weeks.

1. CRITERIA FOR JUDGEMENT (Mark "Yes" or "No").

General statements

What is this work about? In silico replacement therapy for Zoledranate possibly ZINCID13678562

Does it add any value to current knowledge? Very dubious if it does whats wrong with Zoledranate?

Is it innovative? Cannot judge

Yes/No answers.

Is the work scientifically sound? Y

Is the work an original contribution? N
Are the conclusions justified on the evidence presented? NA
Is the work free of major errors in fact, logic or technique? Y
Is the paper clearly and concisely written?Y
Do you consider that the data provided on the care and use of animals (See Instructions to Contributors) is sufficient to establish that the animals used in the experiments were well looked after, that care was taken to avoid distress, and that there was no unethical use of animals? AN ANIMAL ETHICS STATEMENT FROM THE INSTITUTION IS REQUIRED IN TEXT

2. PRESENTATION (Mark "Yes" or "No").

Does the title clearly indicate the content of the paper? No
Does the abstract convey the essence of the article? N see below
Are all the tables essential? Y
Are the figures and drawings of good quality?No
Are the illustrations necessary for an understanding of the text? Y
Is the labelling adequate? Y

3. RECOMMENDATIONS(Mark one with an X)

Not suitable for publication in the OJB
Reassess after major changes X
Reassess after suggested changes X
Accept for publication with minor changes
Accept for publication without changes

4. REPORT This is a theoretical paper describing in silico docking potential for certain compounds able to improve on Zoledronate for the treatment of progeria. The authors should have mentioned why they thought Zoledronate was a problem and why it needs improvement. One reason for publishing this work is the knowledge about Progeria, its current therapy status and drug receptor/docking configuration and application of a bioinformatic solution. Why is the method used innovative? Please explain.. The paper is poorly written with defective grammar throughout. However, the Introduction is well referenced and relevant. In methods the compounds selected from the zinc database appropiately docked on protein FPPS through GLIDE and their ADME score using QikProp. Results were further filtered to obtain ZINCID13678562, The selection process ADME etc needs to be justified (why?) PDF tables and figures not very good quality. Suggest authors prepare whole article in WORD ONLY. Return to authors for major reassessment suggested changes below re english

To be changed see below ABSTRACT

Progeria also called Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by

defective lamin A protein which remain farnesylated after post translational

modification. The mutant protein remain incorporated in the nuclear lamina

leading to mechanical defects, thickening of the nuclear lamina, nuclear lobulation,

and loss of peripheral heterochromatin. Inhibition of protein farnesylation has been

shown to improve abnormal nuclear morphology and phenotype in cellular and

animal models of HGPS. Zoledronate is a potent farnesyl pyrophosphate synthase

inhibitor and is used in the treatment of Progeria (a rare segmental ageing

syndrome). Zoledronate binds to an aspartate-rich region of the enzyme. An

allosteric non-bisphosphonate binding site has been discovered where we found the

zoledronate and its analogs binding effectively. This allosteric site is different from

the active site of the enzyme. Non-bisphosphonate FPPS inhibitor binds to this site.

Total 45 compounds were selected from the Zinc database were docked on protein

FPPS through GLIDE and their ADME score were calculated from QikProp and

analysed. The compound ZINC 13678562 has shown the GLIDE Score of -7.97

and ADME score in the range in which 95% drug lies, which is best among all the

analogs selected for the study. This compounds bind in the same domain where the

Zoledronate molecule binds. So, this compound can be the potent drug molecule

for the treatment of Progeria and can replace the Zoledronate molecule for the

treatment of this disease.

Keywords: - Progeria, Zoledronate, GLIDE, ADME, FPPS.

Changed to:. Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by defective lamin A protein which remains farnesylated after post translational modification. Zoledronate is a potent farnesyl pyrophosphate synthase inhibitor. A total of 45 compounds selected from the Zinc database were docked on protein FPPS through GLIDE and their ADME score were calculated from QikProp. Eighteen compounds had ADME scores and 5 of them had a greater negative GLIDE score than the reference Zoledronate. The analysis suggests that ZINCID13678562, could be used as an alternative to Zoledranate if confirmed by In Vivo pharmacology.

INTRODUCTION

Progeria (Hutchinson-Gilford Progeria syndrome) is a rare segmental aging

syndrome that offers considerable insight into the biology of premature aging.

Scientifically the word Progeria comes from greek word pro means before and

geros means old age. It is the global accelerated cardiovascular and

cerebrovascular diseases that results in premature death between the ages of 7 and

20 years. It has an incidence of 1 in 4,000,000 live births [1]. HGPS is

characterized by retarded growth, partial lipodystrophy, osteoporosis, osteolytic

lesions, thin skin, micrognathia and premature atherosclerosis [2].Different forms

of Progeria are caused by a defect in the conversion of farnesyl-prelamin A to

mature lamin A, which acts as a key structural protein of the nuclear lamina.

Most HGPS patients have a single-nucleotide substitution, at position 1824 C > T,

in the LMNA gene, which activates a cryptic splice donor, resulting in a mutant

mRNA that is translated into a lamin A lacking 50 amino acids. The deleted region

includes the second cleavage site in pre – lamin A, which is usually cleaved by the

endoprotease Zmpste24, but in patients, no cleavage occurs, and the lamin A is

permanently farnesylated and carboxmethylated. The mutant protein, which is

termed progerin/LA _ 50, incorporates abnormally into the nuclear lamina, leading

to mechanical defects ,thickening of the nuclear lamina, nuclear lobulation, and

loss of peripheral heterochromatin .It also causes changes in histone modifications

and increased DNA damage as well as a delay in nuclear reassembly, abnormal

chromosome segregation, and binucleated cells[3].

Farnesylation of aberrant lamin A variant may be targeted by drugs acting on the

synthetic pathway of farnesyl pyrophosphate, a cosubstrate of farnesyltransferase

and a precursor of geranyltransferase pyrophosphate, the substrate of

geranylgeranyltransferase I [4].

Zoledronate (aminobisphosphonate) a potent farnesyl pyrophosphate synthase (an

enzyme in the mevalonate pathway) inhibitor. Inhibition of this enzyme prevents the

biosynthesis of isoprenoid lipids (FPP and GGPP) that are essential for post

translation farnesylation and geranylgeranylation of small G [5].

We used the analogues of zoledronate available in the Zinc Database

(www.zinc.docking.org) and docked with the farnesyl pyrophosphate synthase.

The compounds were found to bind to the positively charged ARG60 present in the

druggable site for non-bisphosphonate FPPS inhibitors [1].The results showed few

of them are more potent drugs than zoledronate. We here present our result on the

basis of different parameters analysed from which we can say that these analogs

can also be used as better FPPS inhibitor than zoledronate.

Materials And Methods

Preparation of protein target structure

Docking studies was conducted using the SP docking protocol of GLIDE

(Schrodinger, LLC) in HP system with Intel core2 duo CPU E8300 @ 2.83 GHz

processor, 1 GB RAM and with the operating system Red hat Linux enterprises

(version 4.0). The 3D structure of the protein, Farnesyl Pyrophosphate synthase

(FPPS) (PDB ID: 3N45) was retrieved from the protein data bank (www.rcsb.org)

and further modified by the protein preparation wizard of GLIDE Docking

(Schrodinger, Inc). The ligands were identified and removed from the structure and

the protein was minimized by applying the force field OPLS_2005. Water

molecules were removed (except HOH192, HOH377, HOH382, HOH383,

HOH406, HOH420, and HOH484 present in the binding site) and H-atoms were

added to the structure. The docking grid was generated with the GLIDE Grid

generation module.

Preparation of compounds

45 compounds were picked up from Zinc Database (www.zinc.docking.org) which

was showing atleast 50% similarity to the Zoledronate and having molecular

weight less than 500 g/mol. These compounds were imported in the maestro

window and prepared using the LigPrep module, which were further used for the

docking with the prepared protein structure of FPPS.

Glide Docking and scoring function

GLIDE calculations were performed with version 5.0 (Schrodinger, Inc). It

performs grid-based ligand docking with energetics and searches for favorable

interactions between one or more typically small ligand molecules and a typically

larger receptor molecule, usually a protein. After ensuring that protein and ligand

are in correct form for docking, the receptor-grid files were generated using gridreceptor

generation program. The ligands were docked with the receptor protein

molecule using GLIDE Algorithm. The ligand poses that GLIDE generates pass

through a series of hierarchical filters that evaluate the ligand’s interaction with the

receptors. The initial filters test the spatial fit of the ligand to the defined active

site, and examine the complementarily of ligand-receptor interactions using a grid

based method patterned after the empirical ChemScore function. The final energy

evaluation is done with Glide score (GScore) and a single best pose is generated as

the output for a particular ligand.

GScore = a * vdW + b * Coul + Lipo + Hbond + Metal + BuryP + RotB + Site

where vdW is van der Waals energy; Coul, Coulomb energy; Lipo, lipophilic

contact term; HBond, hydrogen-bonding term; Metal, metal-binding term; BuryP,

penalty for buried polar groups; RotB, penalty for freezing rotatable bonds; Site,

polar interactions in the active site; and the coefficients of vdW and Coul are: a =

0.065, b = 0.130.

The choice of the best docked structure for each ligand was made using model

energy score (Emodel) that combines GLIDE score, the nonbonded interaction

energy and the excess internal energy of the generated ligand conformation.

GLIDE computed the nonbonded interaction energy as a specially constructed

Coulomb-van der Waals interaction-energy score (CvdW) that is formulated to

avoid overlay rewarding charge-charge interactions at the expense of charge dipole

and dipole-dipole interactions. This score is intended to be more suitable for

comparing the binding affinities of different ligands than is the “raw” Coulombvan

der Waals interaction energy.

ADME screening

The QuikProp module was used to study the ADME properties i.e. Absorption,

Distribution, Metabolism and Excretion. It predicts the pharmaceuticals relevant

properties and the principal physical descriptors. The compounds were neutralized

before being used by QuikProp. The program was processed in normal mode,

predicting 48 properties for each of 45 molecules which includes the principal

descriptors and the physiochemical properties like predicted octanol/water partition

coefficient (QPlogPo/w), predicted octanol/gas partition coefficient (QPlogPoct),

predicted water/gas partition coefficient (QPlogPw), predicted polarizability in

cubic angstroms (QPpolrz),% human oral absorption in intestine (QP%), predicted

brain/blood partition coefficient (QPlogBB), predicted IC50 value for blockage of

HERG K+ channels (log HERG), predicted skin permeability (QPlogKp),

prediction of binding to human serum albumin (QPlogKhsa), predicted apparent

Caco-2 cell permeability in nm/sec (QPPCaco) and predicted apparent MDCK cell

permeability in nm/sec (QPPMDCK). Caco-2 cells are a model for the gut-blood

barrier whereas MDCK cells are considered to be a good mimic for the blood-brain

barrier. It also evaluates the acceptability of the analogues based on the Lipinski's

rule of 5 (number of violations of Lipinski’s rule of five) which is essential for

rational drug design. Poor absorption or permeation are more likely when a ligand

molecule violates Lipinski’s rule of five i.e., has more than 5 hydrogen bond

donors, the molecular weight is over 500, the log P is over 5 and the sum of N’s

and O’s is over 10.

RESULTS AND DISCUSSIONS

Interaction of the compounds with the protein, FPPS (PDB ID: 3N45) was studied

by the molecular docking program GLIDE. 45 compounds were docked with the

FPPS protein whose glide score and energy was tabulated in the table No.1 given

underneath.

Table No.1:-Docking score of compounds with the protein FPPS(RCSB:3N45)

SNo. ZINC ID Glide Score EModel

1 ZINC13643028 -8.04 -90.0

2 ZINC13678562 -7.97 -88.2

3 ZINC13643025 -7.85 -87.7

4 ZINC13643229 -7.19 -80.0

5 ZINC40846382 -7.16 -80.1

6 ZINC33818248 -7.12 -80.5

7 ZINC34641298 -7.09 -81.7

8 ZINC35049970 -7.09 -76.9

9 ZINC13643019 -7.03 -80.4

10 ZINC27204470 -6.99 -70.9

11 ZINC35049962 -6.97 -83.8

12 ZINC28475368 -6.87 -75.6

13 ZINC35049969 -6.87 -78.1

14 ZINC19632644(Zoledronate) -6.81 -75.8

15 ZINC13643046 -6.80 -73.8

16 ZINC13678578 -6.79 -73.2

17 ZINC26742421 -6.77 -78.0

18 ZINC13643229 -6.75 -76.7

19 ZINC40762415 -6.72 -70.4

20 ZINC34493975 -6.71 -72.1

21 ZINC26740680 -6.71 -75.1

22 ZINC35049980 -6.68 -76.3

23 ZINC26745617 -6.67 -74.8

24 ZINC35049964 -6.67 -73.3

25 ZINC40980794 -6.66 -70.7

26 ZINC13643058 -6.63 -68.4

27 ZINC13643040 -6.61 -75.3

28 ZINC35049981 -6.61 -74.3

29 ZINC26741243 -6.60 -69.9

30 ZINC34031362 -6.54 -70.0

31 ZINC34639506 -6.53 -70.7

32 ZINC13643055 -6.45 -75.6

33 ZINC35049982 -6.44 -72.8

34 ZINC13643043 -6.41 -78.1

35 ZINC13529694 -6.41 -64.9

36 ZINC13643034 -6.40 -81.2

37 ZINC13643037 -6.40 -79.5

38 ZINC13643022 -6.39 -69.7

39 ZINC40836037 -6.37 -72.6

40 ZINC27300956 -6.21 -68.6

41 ZINC13643031 -6.21 -70.9

42 ZINC13643052 -6.18 -68.9

43 ZINC14950188 -6.04 -65.9

44 ZINC27197056 -5.88 -67.8

45 ZINC13643049 -5.70 -60.5

*The Zinc compound in bold is the reference compound.

The compound with ZINC ID 13643028 has most negative glide score of -8.04 and

Emodel is -90 KJ/mol whereas zoledronate which is the reference compound

having the ZINC ID 19632644 has the glide score of -6.81and Emodel is -88.2

KJ/mol. Among 45 compounds 13 compounds have shown more negative glide

score than Zoledronate.

ADME score of these 45 compounds had been studied by the QuikProp module.

Surprisingly, only 17 compounds has shown the ADME score and the compound

having the most negative glide score have also not shown any ADME score. The

ADME score of the compounds has been tabulated in the Table No.2. Table is

arranged in the ascending order of the Glide score.

ZINC ID MW SASA FOSA FISA PISA WPSA volume QPpolrz QPlogPC16 QPlogPoct QPlogPw QPlogPo/w QPlogS QPLogHERG PHOS GScore

ZINC13678562 348.19 527.781 24.469 251.305 248.09 3.914 928.717 27.604 10.68 18.301 12.485 2.157 -1.445 2.447 36.418 -7.97

ZINC13678563 322.15 462.946 21.514 255.999 181.51 3.927 817.997 22.537 9.429 16.44 11.416 1.623 -0.602 3.079 32.494 -7.19

ZINC13678564 308.13 459.82 30.346 292.828 133.1 3.543 790.242 21.606 8.743 14.76 11.809 -0.214 1.592 3.429 15.487 -7.16

ZINC13678565 306.15 462.314 30.69 242.732 184.9 3.995 808.697 22.839 9.002 13.987 9.364 1.533 0.234 3.328 34.219 -7.12

ZINC13678566 221.04 345.602 33.139 308.841 0 3.621 554.986 10.278 6.395 12.914 10.245 -2.208 0.814 4.082 0 -6.99

ZINC13678567 272.09 407.158 24.561 271.02 108.49 3.088 690.068 16.72 7.875 16.544 11.379 0.515 0.354 4.008 23.457 -6.81

ZINC13678568 286.12 454.446 34.187 287.71 128.46 4.087 761.381 19.122 8.535 17.984 11.522 0.793 -0.16 3.262 22.251 -6.77

ZINC13678569 322.15 474.224 21.103 260.727 188.43 3.959 820.36 22.697 9.396 16.373 11.522 1.595 -0.773 3.079 31.528 -6.75

ZINC13678570 273.08 396.768 31.855 290.933 72.166 1.814 671.348 15.624 7.627 14.103 12.227 -0.142 0.772 4.295 16.227 -6.71

ZINC13678571 286.12 433.094 83.774 261.092 84.898 3.33 736.817 18.365 7.966 17.463 10.721 0.997 -0.156 3.852 27.966 -6.67

ZINC13678572 256.09 409.595 33.882 258.078 113.63 4.01 682.294 17.1 7.411 12.672 9.35 0.275 1.495 3.749 24.25 -6.66

ZINC13678573 286.12 439.676 39.753 275.437 121.57 2.92 743.959 18.359 8.333 14.095 11.356 0.78 0.06 3.262 24.26 -6.6

ZINC13678574 300.14 452.224 156.35 259.543 32.45 3.884 796.204 20.241 8.231 14.886 10.4 1.3 -0.441 4.227 30.003 -6.6

ZINC13678575 286.12 431.51 84.973 257.229 85.339 3.969 734.633 18.282 7.937 16.925 10.694 1.016 -0.136 3.852 28.73 -6.52

ZINC13678576 300.14 449.662 167.75 252.454 26.328 3.13 778.271 19.465 7.893 14.789 10.304 1.227 -0.399 4.116 30.777 -6.48

ZINC13678577 342.57 482.775 30.706 282.227 94.108 75.74 833.402 22.961 9.248 15.488 11.06 0.614 0.581 3.423 22.136 -6.4

ZINC13678578 273.08 403.285 25.734 293.482 81.282 2.787 679.945 16.055 7.736 14.88 11.83 0.081 0.546 4.133 17.104 -6.21

ZINC13678579 256.09 407.673 34.83 254.319 114.52 4.003 675.526 16.837 7.306 12.319 9.325 0.258 1.531 3.749 24.788 -6.06

MW- Molecular weight (Range:- 135-175 g/mol), SASA- Total solvent accessible surface area (Range:- 3000-1000 Å2), FOSA- Hydrophobic component of the

SASA (Range- 0-750 Å2), FISA- Hydrophilic component of the SASA (Range:- 7-330 Å2), PISA- ¼ component of the SASA (Range:- 0-400 Å2), WPSA- Weakly

component of SASA (Range:- 0-150 Å2) , Volume- Total solvent accessible area (Range:- 500-2000 Å3), QPpolrz- Predicted polarizability (Range:- 13-70 Å3 ),

QPlogPC16- Predicted log of hexadecane/gas partition coefficient (Range:- 4-18), QPlogPoct- Predicted log of octanol/gas partition coefficient (Range:- 8-43),

QPlogPw- Predicted log of water/gas partition coefficient (Range:- 5-48), QPlogPo/w- Predicted log of octanol/water partition coefficient (Range:- -2-6),

QplogS- Predicted log of aqueous solubility ( Range:- -6-0.5 mol/L), QPlogHERG- Predicted IC50 value for blockage of HERG K+ channels (Range:- concrern

below -5), PHOS- Predicted Human Oral Absorption, GScore- Glide Score(http://ccc.chem.pitt.edu/UPCMLD/Scaffolds/QP%202D.pdf ).The valued in red are out

of range from the category of Drug and the first Zinc compounds in bold is showing best ADME and dock score whereas the second compound in bold is the

reference compound.

Table 2:- ADME Scores

The compounds which are having more negative glide sore than zoledronate and

shown the ADME score were selected for the study and the rest compounds were

escaped. The number of compound satisfying these criteria was found to be 5. The

properties depicted by the QikProp module were analyzed with the reference score

of each property. The compound with ZINCID13678562 is showing all the

property in the range in which 95% of drugs lies and have the second most

negative glide score of -7.97 and EModel -88.2 KJ/mol. The molecular weight of

this compound is 348.188g/mol whereas zoledronate is having 272.091g/mol little

bit high but it is in the range i.e. 130g/mol-725g/mol. The values of Total solvent

accessible surface area (SASA), Hydrophobic component of the SASA (saturated

carbon and attached hydrogen) i.e. FOSA, Hydrophilic component of the SASA

(N, O and H on heteroatom) i.e. FISA, Weakly polar component of the SASA

(halogens, P, S) i.e. WPSA are 527.781Å2, 24.469 Å2, 251.305 Å2, 3.914 Å2

respectively. The predicted polarizability (QPpolrz), predicted log of octanol/gas

partition coefficient (QPlogPoct), predicted log of water/gas partition coefficient

(QPlogPw), predicted log of octanol/water partition coefficient (QPlogPo/w),

predicted log of aqueous solubility (QPlogS), predicted IC50 value for blockage of

HERG K+ channels(QPlogHERG) are 27.604 Å3, 18.301, 12.485, 2.157, -1.445

mol/L, 2.447 respectively. The percentage human oral absorption of this compound

is 36.418%. (http://ccc.chem.pitt.edu/UPCMLD/Scaffolds/QP%202D.pdf). The

compound ZINC13643229 and ZINC33818248 having the docking score of -7.19

and -7.12 respectively are also showing all the properties in the range in which the

95% of drug lies but the predicted log of aqueous solubility (QPlogS), of the

compound ZINC40846382 is not in the range and its value is exceeding by 1.092

whereas the predicted polarizability (QPpolrz), predicted log of octanol/water

partition coefficient (QPlogPo/w), and predicted log of aqueous solubility

(QPlogS) of the compound ZINC27204470 are out of range. Even the Percentage

human oral absorption is 0% for this compound. All the ADME properties of the

reference compound ZINC19632644 (Zoledronate) is in the range.

On critical investigation of the binding pattern of the compound ZINCID13678562

it is observed this exhibits binding to the same domain where the zoledronate

binds.

Figure 1:- Docked compound ZINCID13678562 with protein FPPS

The terminal phosphate group binds with the amino acid Arginine (ARG60) and

water molecule (HOH382). The oxygen molecule of the other phosphate group

binds with the water molecules (HOH420 and HOH484). The hydroxyl of this

compound binds with another water molecule (HOH392).

Figure 2:- Docked compound Zoledronate with protein FPPS

The compound zoledronate on other hand binds with Arginine (ARG60) through

the terminal phosphate group. The other phosphate group binds with the water

molecule (HOH420 and HOH484) whereas the Hydroxyl group is showing bonds

with the water molecule (HOH392). Zoledronate has been reported to bind to the

aspartate-rich region of the enzyme via chelation with three Mg2+. This region is

found in the highly conserved domain II and VI of FPPS [6]. An allosteric binding

pocket near the C terminus of the enzyme and adjacent to the isopentenyl

pyrophosphate (IPP) binding site has been discovered where non-bisphosphonate

FPPS inhibitors bind which is reported to be druggable site. The pocket is mainly

lined by helices áC, áG, áH and the C-terminal helix áJ. In addition, few residues

from the áB-áC loop, the áH-áI loop, the N-terminal helix áA and the C-terminal

loop are also contributing in some of the complexes. The pocket comprises a

hydrophobic base and rear side, involving the side chains of Phe206, Phe239,

Leu344, Ile348 and Tyr10. In sharp contrast, the opposite side comprises several

positively charged side chains (Lys57, Arg60, Lys347) as well as polar side chains

(Asn59, Thr63) [1].

Conclusion

In the present study it is predicted that out of the 45 compounds selected for the

study for the inhibition of FPPS, only 18 compounds have shown the ADME

scores and out of these only 5 compounds have shown more negative GLIDE score

than the reference compound Zoledronate. Among these 5 compounds

ZINCID13678562, ZINC13643229 and ZINC33818248 are showing all the

ADME scores in the range in which the 95% of drug lies and more negative

GLIDE score than Zoledronate. Among these three the compound

ZINCID13678562 have the most negative GLIDE score and can therefore be used

as an alternative for the Zoledranate. Thus the study confirm, the compound

ZINCID13678562 can act as a drug against the treatment of Progeria yet

Pharmacological study will confirm it to be promising.

REFERENCES

1 Mark W. Kieran et.al (2007); New Approaches to progeria.Pediatrics.120, 834-

841.

2. Julia I. Toth et.al (2005); Blocking protein farnesyltransferase improves nuclear

shape in fibroblasts from humans with progeroid syndromes .PNAS.102, 12873-

12878.

3. Eran Meshorer; Yosef Gruenbaum; Gone with the Wnt/Notch:stem cells in

laminopathies,progeria, and aging.The Journal of Cell Biology.181,9-13(2008).

4. Wolfgang Jahnke et.al (2010); Allosteric non-bisphosphonate FPPS inhibitors

identified by fragment-based discovery; nature chemical biology.6, 661-664.

5. Claire L. Navarro et.al (2006); Molecular bases of progeroid syndromes.Human

Molecular Genetics. 15,151-161

6. Andrea Montalvetti et.al (2001); Bisphosphonates are potent inhibitors of

Trypanosoma cruzi Farnesyl Pyrophosphate Synthase.J Biol Chem.276, 33930-

33937.

ACKNOWLEDGEMENT

The authors are thankful to the Sub-Distributed Information Center (BTISnet

SubDIC), Department of Biotechnology (No. BT/BI/04/065/04), New Delhi, India

and the Government of Jharkhand, Dept. of Agriculture for providing

infrastructure development fund for this department. We are highly

acknowledged the kind support of the Department of Pharmaceutical Sciences,

Birla Institute of Technology Mesra, for providing softwares and computer

facilities.

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Suggested article

NEW TITLE Inhibition of Farnesyl pyrophosphate by allosteric site drug binding

ABSTRACT

Kumar S, Pathak J, Asha PJ, Bandopadhyay R, Inhibition of Farnesyl pyrophosphate by allosteric site drug binding, Online J Bioinformatics, 12(1):57-65, 2011 . Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by defective lamin A protein which remains farnesylated after post translational modification. Zoledronate is a potent farnesyl pyrophosphate synthase inhibitor. A total of 45 compounds selected from the Zinc database were docked on protein FPPS through GLIDE and their ADME score were calculated from QikProp. Eighteen compounds had ADME scores and 5 of them had a greater negative GLIDE score than the reference Zoledronate. The analysis suggests that ZINCID13678562, could be used as an alternative to Zoledranate if confirmed by In Vivo pharmacology.

INTRODUCTION

Re-Write correct grammar throughout Progeria (Hutchinson-Gilford Progeria syndrome) is a rare segmental aging syndrome that offers considerable insight into the biology of premature aging. Scientifically the word Progeria comes from greek word pro means before and geros means old age. It is the global accelerated cardiovascular and cerebrovascular diseases that results in premature death between the ages of 7 and 20 years. It has an incidence of 1 in 4,000,000 live births [1]. HGPS is characterized by retarded growth, partial lipodystrophy, osteoporosis, osteolytic lesions, thin skin, micrognathia and premature atherosclerosis [2]. Different forms of Progeria are caused by a defect in the conversion of farnesyl-prelamin A to mature lamin A, which acts as a key structural protein of the nuclear lamina.

Most HGPS patients have a single-nucleotide substitution, at position 1824 C > T, in the LMNA gene, which activates a cryptic splice donor, resulting in a mutant mRNA that is translated into a lamin A lacking 50 amino acids. The deleted region includes the second cleavage site in pre – lamin A, which is usually cleaved by the endoprotease Zmpste24, but in patients, no cleavage occurs, and the lamin A is permanently farnesylated and carboxmethylated. The mutant protein, which is termed progerin/LA _ 50, incorporates abnormally into the nuclear lamina, leading to mechanical defects ,thickening of the nuclear lamina, nuclear lobulation, and loss of peripheral heterochromatin. The condition also causes changes in histone modifications and increased DNA damage as well as a delay in nuclear reassembly, abnormal chromosome segregation, and binucleated cells[3].

Farnesylation of aberrant lamin A variant may be targeted by drugs acting on the synthetic pathway of farnesyl pyrophosphate, a cosubstrate of farnesyltransferase and a precursor of geranyltransferase pyrophosphate, the substrate of geranylgeranyltransferase I [4]. oledronate (aminobisphosphonate) a potent farnesyl pyrophosphate synthase (an enzyme in the evalonate pathway) inhibitor. Inhibition of this enzyme prevents the biosynthesis of isoprenoid lipids (FPP and GGPP) that are essential for post translation farnesylation and geranylgeranylation of small G [5].

We used the analogues of zoledronate available in the Zinc Database(www.zinc.docking.org) and docked with the farnesyl pyrophosphate synthase. The compounds were found to bind to the positively charged ARG60 present in the druggable site for non-bisphosphonate FPPS inhibitors [1].The results showed few of them are more potent drugs than zoledronate. We here present our result on the basis of different parameters analysed from which we can say that these analogs can also be used as better FPPS inhibitor than zoledronate. Re write not acceptable

MATERIALS AND METHODS

Protein target structure

Docking studies was conducted using the SP docking protocol of GLIDE (Schrodinger, LLC) in HP system with Intel core2 duo CPU E8300 @ 2.83 GHz processor, 1 GB RAM and with the operating system Red hat Linux enterprises (version 4.0). The 3D structure of the protein, Farnesyl Pyrophosphate synthase (FPPS) (PDB ID: 3N45) was retrieved from the protein data bank (www.rcsb.org) and further modified by the protein preparation wizard of GLIDE Docking (Schrodinger, Inc). The ligands were identified and removed from the structure and the protein was minimized by applying the force field OPLS_2005. Water molecules were removed (except HOH192, HOH377, HOH382, HOH383, HOH406, HOH420, and HOH484 present in the binding site) and H-atoms were added to the structure. The docking grid was generated with the GLIDE Grid generation module.

Compounds

45 compounds were picked up from Zinc Database (www.zinc.docking.org) which was showing atleast 50% similarity to the Zoledronate and having molecular weight less than 500 g/mol. These compounds were imported in the maestro window and prepared using the LigPrep module, which were further used for the docking with the prepared protein structure of FPPS.

Glide Docking and scoring function

GLIDE calculations were performed with version 5.0 (Schrodinger, Inc). T he program performs grid-based ligand docking with energetics and searches for favorable interactions between one or more typically small ligand molecules and a typically larger receptor molecule, usually a protein. After ensuring that protein and ligand are in correct form for docking, the receptor-grid files were generated using gridreceptor generation program. The ligands were docked with the receptor protein molecule using GLIDE Algorithm. The ligand poses that GLIDE generates pass

through a series of hierarchical filters that evaluate the ligand’s interaction with the receptors. The initial filters test the spatial fit of the ligand to the defined active site, and examine the complementarily of ligand-receptor interactions using a grid based method patterned after the empirical ChemScore function. The final energy evaluation is done with Glide score (GScore) and a single best pose is generated as the output for a particular ligand.

GScore = a * vdW + b * Coul + Lipo + Hbond + Metal + BuryP + RotB + Site where vdW is van der Waals energy; Coul, Coulomb energy; Lipo, lipophilic contact term; HBond, hydrogen-bonding term; Metal, metal-binding term; BuryP, penalty for buried polar groups; RotB, penalty for freezing rotatable bonds; Site, polar interactions in the active site; and the coefficients of vdW and Coul are: a = 0.065, b = 0.130. The choice of the best docked structure for each ligand was made using model energy score (Emodel) that combines GLIDE score, the nonbonded interaction energy and the excess internal energy of the generated ligand conformation. GLIDE computed the non bonded interaction energy as a specially constructed Coulomb-van der Waals interaction-energy score (CvdW) that is formulated to avoid overlay rewarding charge-charge interactions at the expense of charge dipole and dipole-dipole interactions. This score is intended to be more suitable for comparing the binding affinities of different ligands than is the “raw” Coulombvan der Waals interaction energy.

ADME screening

The QuikProp module was used to study the ADME properties i.e. Absorption, Distribution, Metabolism and Excretion. It predicts the pharmaceuticals relevant properties and the principal physical descriptors. The compounds were neutralizedbefore being used by QuikProp. The program was processed in normal mode, predicting 48 properties for each of 45 molecules which includes the principal descriptors and the physiochemical properties like predicted octanol/water partition coefficient (QPlogPo/w), predicted octanol/gas partition coefficient (QPlogPoct), predicted water/gas partition coefficient (QPlogPw), predicted polarizability in cubic angstroms (QPpolrz),% human oral absorption in intestine (QP%), predicted brain/blood partition coefficient (QPlogBB), predicted IC50 value for blockage of HERG K+ channels (log HERG), predicted skin permeability (QPlogKp), prediction of binding to human serum albumin (QPlogKhsa), predicted apparent Caco-2 cell permeability in nm/sec (QPPCaco) and predicted apparent MDCK cell permeability in nm/sec (QPPMDCK). Caco-2 cells are a model for the gut-blood barrier whereas MDCK cells are considered to be a good mimic for the blood-brain barrier. It also evaluates the acceptability of the analogues based on the Lipinski's rule of 5 (number of violations of Lipinski’s rule of five) which is essential for rational drug design. Poor absorption or permeation are more likely when a ligand molecule violates Lipinski’s rule of five i.e., has more than 5 hydrogen bond donors, the molecular weight is over 500, the log P is over 5 and the sum of N’s and O’s is over 10.

RESULTS AND DISCUSSIONS

Interaction of the compounds with the protein, FPPS (PDB ID: 3N45) was determined using GLIDE (see methods). 45 compounds were docked with the FPPS protein whose glide score and energy was tabulated on Table 1 (next page).

The compound ZINC ID 13643028 had the greatest negative glide score of -8.04 and Emodel is -90 KJ/mol whereas zoledronate which is the reference compound ZINC ID 19632644 has a score of -6.81 and Emodel, -88.2 KJ/mol. Amongst the 45 compounds, 13 had scores more negative than Zoledronate, 17 had ADME scores but the compound with the greatest negative glide did not a ADME score. The ADME score of the compounds is shown in Table 2. The data is arranged in an ascending Glide score order.

Table 2:- ADME Scores

MW- Molecular weight (Range:- 135-175 g/mol), SASA- Total solvent accessible surface area (Range:- 3000-1000 Å2), FOSA- Hydrophobic component of the SASA (Range- 0-750 Å2), FISA- Hydrophilic component of the SASA (Range:- 7-330 Å2), PISA- ¼ component of the SASA (Range:- 0-400 Å2), WPSA- Weakly component of SASA (Range:- 0-150 Å2) , Volume- Total solvent accessible area (Range:- 500-2000 Å3), QPpolrz- Predicted polarizability (Range:- 13-70 Å3 ), QPlogPC16- Predicted log of hexadecane/gas partition coefficient (Range:- 4-18), QPlogPoct- Predicted log of octanol/gas partition coefficient (Range:- 8-43), QPlogPw- Predicted log of water/gas partition coefficient (Range:- 5-48), QPlogPo/w- Predicted log of octanol/water partition coefficient (Range:- -2-6), QplogS- Predicted log of aqueous solubility ( Range:- -6-0.5 mol/L), QPlogHERG- Predicted IC50 value for blockage of HERG K+ channels (Range:- concern below -5), PHOS- Predicted Human Oral Absorption, GScore- Glide Score(http://ccc.chem.pitt.edu/UPCMLD/Scaffolds/QP%202D.pdf ).The valued in red are out of range from the category of Drug and the first Zinc compounds in bold is showing best ADME and dock score whereas the second compound in bold is the reference compound

The compounds which had a greater negative score than zoledronate and had an ADME score were selected. The number of compound satisfying these criteria was found to be 5. The properties depicted by the QikProp module were analyzed with the reference score of each property. The compound with ZINCID13678562 is showing all the property in the range in which 95% of drugs lies and have the second most negative glide score of -7.97 and EModel -88.2 KJ/mol. The molecular weight of this compound is 348.188g/mol whereas zoledronate is having 272.091g/mol little bit high but it is in the range i.e. 130g/mol-725g/mol. The values of Total solvent accessible surface area (SASA), Hydrophobic component of the SASA (saturated carbon and attached hydrogen) i.e. FOSA, Hydrophilic component of the SASA(N, O and H on heteroatom) i.e. FISA, Weakly polar component of the SASA(halogens, P, S) i.e. WPSA are 527.781Å2, 24.469 Å2, 251.305 Å2, 3.914 Å2 respectively. The predicted polarizability (QPpolrz), predicted log of octanol/gas partition coefficient (QPlogPoct), predicted log of water/gas partition coefficient(QPlogPw), predicted log of octanol/water partition coefficient (QPlogPo/w),predicted log of aqueous solubility (QPlogS), predicted IC50 value for blockage of HERG K+ channels(QPlogHERG) are 27.604 Å3, 18.301, 12.485, 2.157, -1.445mol/L, 2.447 respectively. The percentage human oral absorption of this compoundis 36.418%. (http://ccc.chem.pitt.edu/UPCMLD/Scaffolds/QP%202D.pdf). The compound ZINC13643229 and ZINC33818248 having the docking score of -7.19and -7.12 respectively are also showing all the properties in the range in which the95% of drug lies but the predicted log of aqueous solubility (QPlogS), of thecompound ZINC40846382 is not in the range and its value is exceeding by 1.092 whereas the predicted polarizability (QPpolrz), predicted log of octanol/water partition coefficient (QPlogPo/w), and predicted log of aqueous solubility (QPlogS) of the compound ZINC27204470 are out of range. Even the Percentage human oral absorption is 0% for this compound. All the ADME properties of the reference compound ZINC19632644 (Zoledronate) is in the range.

On critical investigation of the binding pattern of the compound ZINCID13678562 it is observed this exhibits binding to the same domain where the zoledronate binds.

The terminal phosphate group binds with the amino acid Arginine (ARG60) and water molecule (HOH382). The oxygen molecule of the other phosphate group binds with the water molecules HOH420 and HOH484). The hydroxyl of this compound binds with another water molecule (HOH392).

Figure 2:- Docked compound Zoledronate with protein FPPS

The compound zoledronate on other hand binds with Arginine (ARG60) through the terminal phosphate group. The other phosphate group binds with the water molecule (HOH420 and HOH484) whereas the Hydroxyl group has a bond with the water molecule (HOH392). Zoledronate has been reported to bind to the aspartate-rich region of the enzyme via chelation with three Mg2+. This region is found in the highly conserved domain II and VI of FPPS [6]. An allosteric binding pocket near the C terminus of the enzyme and adjacent to the isopentenyl pyrophosphate (IPP) binding site has been discovered where non-bisphosphonate FPPS inhibitors bind which is reported to be druggable site. The pocket is mainly lined by helices áC, áG, áH and the C-terminal helix áJ. In addition, few residues from the áB-áC loop, the áH-áI loop, the N-terminal helix áA and the C-terminal loop are also contributing in some of the complexes. The pocket comprises a hydrophobic base and rear side, involving the side chains of Phe206, Phe239, Leu344, Ile348 and Tyr10. In sharp contrast, the opposite side comprises several positively charged side chains (Lys57, Arg60, Lys347) as well as polar side chains

(Asn59, Thr63) [1].

CONCLUSION

From 45 compounds used to evaluate In Silico inhibition of FPPS, only 18 had ADME scores and 5 had greater negative GLIDE score than Zoledronate. ZINCID13678562 had the greater negative GLIDE score.

ACKNOWLEDGEMENT

The authors are thankful to the Sub-Distributed Information Center (BTISnet SubDIC), Department of Biotechnology (No. BT/BI/04/065/04), New Delhi, India and the Government of Jharkhand, Dept. of Agriculture for providing infrastructure development fund for this department. We are highly acknowledged the kind support of the Department of Pharmaceutical Sciences, Birla Institute of Technology Mesra, for providing softwares and computer facilities.

REFERENCES

1. Mark W. Kieran et.al (2007); New Approaches to progeria. Pediatrics.120, 834-841.

2. Julia I. Toth et.al (2005); Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes .PNAS.102, 12873-12878.

3. Eran Meshorer; Yosef Gruenbaum; Gone with the Wnt/Notch:stem cells in laminopathies,progeria, and aging.The Journal of Cell Biology.181,9-13(2008).

4. Wolfgang Jahnke et.al (2010); Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery; nature chemical biology.6, 661-664.

5. Claire L. Navarro et.al (2006); Molecular bases of progeroid syndromes.Human

Molecular Genetics. 15,151-161

6. Andrea Montalvetti et.al (2001); Bisphosphonates are potent inhibitors of Trypanosoma cruzi Farnesyl Pyrophosphate Synthase.J Biol Chem.276, 33930-33937.

REPLY received 28/3/11

Answers to the Questions raised by the reviewer
Qno.1:- Why Zoledronate is a Problem and why it needs replacement?
Ans:- The major drawback with the zoledronate is that it is poorly absorbed, oral absorption is about 1% of what intravenous absorption. That’s why it needs replacement.
Qno. 2:- Why is the method used innovative?
Ans:- Totally can’t say the method used is innovative because this is the general process for the finding the substitute for the existing drug for its better effect. Here, we also tried to substitute the zoledronate with the better compound. We picked compounds from Zinc Database and studied its docking and ADME properties.
Qno.3:- The selection process ADME etc needs to be justified (why?)
Ans:- Through the ADME score we can predict the pharmacological properties of compounds. The Quikprop program predicts around 48 properties associated with its pharmacology. We have selected the important pharmacological properties and escaped the other properties like violation of Lipinski rule, Rule of 3, no of amide groups, dipole moments, ionization potential, electron affinity etc. After selecting the properties on which screening has to be done, we looked for the range, and screened the compounds.