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Biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-methylamide (CA224), a non-planar analog of fascaplysin inhibits Cdk4 and tubulin polymerization: Evaluation of in vitro and in vivo anticancer activity Sachin Mahale, Sandip Bibishan Bharate, sudhakar manda, prashant joshi, Sonali S. Bharate, Paul R Jenkins, Ram A. Vishwakarma, and Bhabatosh Chaudhuri J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5014743 • Publication Date (Web): 04 Nov 2014 Downloaded from http://pubs.acs.org on November 8, 2014

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Journal of Medicinal Chemistry

Biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]methylamide (CA224), a non-planar analog of fascaplysin inhibits Cdk4 and tubulin polymerization: Evaluation of in vitro and in vivo anticancer activity Sachin Mahale, ‡, Sandip B. Bharate,*, †,ǁ Sudhakar Manda,†,ǁ Prashant Joshi,†, ǁ Sonali S. Bharate,⊥ Paul R. Jenkins,∞ Ram A. Vishwakarma,*, †,ǁ Bhabatosh Chaudhuri*,‡ ‡

School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK

†

Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-

180001, India ǁ

Academy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine,

Canal Road, Jammu-180001, India ⊥Preformulation

Laboratory, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-

180001, India ∞

Department of Chemistry, University of Leicester, Leicester, LE1 7RH, UK

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ABSTRACT Biphenyl-4-carboxylic acid-[2-(1H-indol-3-yl)-ethyl]-methylamide 1 (CA224) is a non-planar analog of fascaplysin (2) that specifically inhibits Cdk4-cyclin D1 in-vitro. Compound 1 blocks growth of cancer cells at G0/G1 phase of the cell cycle. It also blocks at G2/M phase which is explained by the fact that it inhibits tubulin polymerization. Besides, it acts as an enhancer of depolymerization for taxol-stabilized tubulin. Western-blot analyses of p53-positive cancer cells treated with compound 1 indicated upregulation of p53, p21 and p27 proteins together with down-regulation of cyclin B1 and Cdk1. Compound 1 selectively induces apoptosis in SV40 large T-antigen transformed cells and significantly reduces colony formation efficiency, in a dose-dependent manner of lung cancer cells. It is efficacious at 1/10th the MTD, against human tumors derived from HCT-116 and NCI-H460 cells in SCID mice models. The promising efficacy of compound 1 in human xenograft models with an excellent therapeutic-window indicates its potential for clinical development.

KEYWORDS Cdk4-cyclin D1, tubulin polymerization, fascaplysin, anti-cancer, chemotherapeutic, cell division cycle


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INTRODUCTION The mammalian cell division is a highly controlled process and the loss of this control results in development of cancer phenotype. Mammalian cells undergo various stages of the cell cycle (G0, G1, S, G2, M-phase), and the transition of cells from one phase to another involves various check-point mechanisms including cyclin-dependent kinases, checkpoint kinases, and their partner cyclins. Malfunctioning of any of these checkpoint mechanisms leads to uncontrolled cell division and proliferation.1-5 Cyclin-dependent kinase 4 and its cyclin partner D1 (check-point mechanism) controls transition of the cells from G1 to S phase of the cell cycle.6-9 Active Cdk4-cyclin D complexes inactivate retinoblastoma protein (pRb) by phosphorylating specifically at Ser780 and Ser795 residues, which allows the G1/S transition of cells during the cell cycle.6, 10 Hyperphosphorylation of pRb leads to loss of control over gene transcription through the E2F family of transcription factors, which ultimately turns into uncontrolled cell division. In contrast, pRb phosphorylation by Cdk2 or Cdk3 along with their cyclin partners A or C is not sufficient to inactivate pRb functions.11-13 A large number of human tumors (around 96%) lose normal cell cycle transition check-point mechanism due to variety of genetic and biochemical adaptations including the hyperactivity of the Cdk4 protein, down-regulation of Cdk4 positive regulator p16INK4A and mutations in pRb. The vital role of Cdk4-cyclin D1 in tumor development has been confirmed by experiments in Cdk4 and cyclin D1 knock-out mice which have been shown to be resistant to tumor development. On the other hand, antisense nucleotides based targeting of cyclin D decreases tumor size and growth in-vivo. Based on these experimental observations, Cdk4-cyclin D complex protein is considered as a promising target for combating cancer.14-16 Inhibition of Cdk4-cyclin D1 with small molecules has been an area of major interest in the field of anticancer drug discovery since last two decades. There have been numerous scientific reports highlighting the role of Cdk4-cyclin D1 inhibitors in cancer treatment.17-22 Pfizer's palbociclib (6-acetyl8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one; ACS Paragon Plus Environment

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PD-0332991), a selective inhibitor of Cdk4 and Cdk6 has received FDA approval for treatment of patients with breast cancer.23,

24

Other Cdk4/Cdk6 dual inhibitors LY2835219 (N-[5-[(4-ethyl-1-

piperazinyl)methyl]-2-pyridinyl]-5-fluoro-4-[4-fluoro-2-methyl-1-(1-methylethyl)-1H-benzimidazol-6yl]-2-pyrimidinamine methanesulfonate) and LEE011 (7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide) are in phase I and III clinical studies for breast cancer (Source: http://clinicaltrials.gov).25, 26 The main objective of our studies was to develop potent and specific small molecule inhibitors of Cdk4-cyclin D1 based on the structure of fascaplysin (2) which is a pentacyclic quaternary salt originally isolated from the Fijian sponge Fascaplysinopsis Bergquist sp.27 It is known to possess antimicrobial, antimalarial and anti-acetylcholinesterase activities.27-29 It also displays potent cytotoxicity against small cell lung cancer cells and induces cell cycle arrest in G0/G1 at lower and Sphase at higher concentrations, respectively.30 Compound 2 also showed anti-tumor effects in sarcoma mice model through apoptotic and anti-angiogenesis pathways.31 Fascaplysin is one of the specific inhibitor of Cdk4-cyclin D1 with IC50 value of 410 nM.9, 32 It inhibits Cdk4-cyclin D1 in-vitro and blocks the growth of normal and cancer cells at the G0/G1 phase of the cell cycle, which correlates with the accumulation of hypo-phosphorylated pRb, implying there is no phosphorylation at Cdk4-specific serine residues.9 Compound 2 has also been reported to inhibit Cdc25B with an IC50 of 1 µg/ml (3.26 µM).33, 34 An analogue of fascaplysin 1-deoxysecofascaplysin A, has been reported to inhibit cell growth of breast cancer MCF-7 and ovarian cancer OVCAR-3 cell lines in-vitro.35 Nonetheless, it is unlikely that fascaplysin will ever be used therapeutically as an anticancer agent because it is highly toxic. The potential for its planar structure to intercalate with double-stranded DNA has been suggested as a possible explanation for its unusual biological activity and toxicity. The DNA binding property of fascaplysin is similar to structurally related DNA intercalating agents like cryptolepine and ellipticine.36 We have explored the possibility of separating the DNA intercalating ability of fascaplysin from its potent Cdk4-specific inhibitory activity. The aim of the current study was ACS Paragon Plus Environment

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therefore to devise non-planar (non-toxic) Cdk4-cyclin D1 inhibitors based on the structure of fascaplysin.37-40 Biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-methylamide 1 (CA224)41 was initially identified as a Cdk4-cyclin D1 specific inhibitor lacking the ability to intercalate DNA. As expected, compound 1 manifested its Cdk4-specific inhibitory ability by blocking cancer cells at the G0/G1 phase of the cell cycle. Surprisingly however, compound 1 also inhibited the G2/M phase in a Cdkindependent manner. Further investigations showed that the compound 1 inhibited the growth of a panel of ten cancer cell lines (some of them potentially resistant to cancer chemotherapy) at a low micromolar range. The validity of these results was corroborated by the observation that compound 1 has the ability to diminish the colony formation efficiency of cancer cells. The Cdk-independent G2/M block was found to be associated with its anti-tubulin activity. Compound 1 inhibited the polymerization of tubulin in-vitro and also showed an enhancing effect on tubulin depolymerization in live cells. In human xenograft models, compound 1 was found to inhibit the growth of HCT-116 and H-460 tumor growth at 1/10th of the MTD, 100 mg/kg. Here, we present the in vitro and in-vivo anticancer activity of compound 1 in detail. RESULTS AND DISCUSSION Chemistry. Our efforts towards discovery of non-toxic analogs of fascaplysin led to the identification of compound 1, a non-planar analog of fascaplysin (2). Briefly, the synthesis of compound 1 involves three steps starting from commercially available tryptamine (3). The treatment of tryptamine (3) with ethyl chloroformate produced carbamate ester 4, which further on reduction using LAH led to formation of N-methyl tryptamine 5. Compound 5 on reaction with 4-biphenyl carbonyl chloride (6) produced compound 1. The detailed lead optimization and SAR have been described in previous publications.37-41 The synthetic scheme for synthesis of compound 1 is shown in Scheme 1. It was interesting to note that the non-planar analogs with opened C and D rings of fascaplysin (2), led to loss of DNA intercalation activity. Among C and D-ring opened analogs, compounds with para-substituted ACS Paragon Plus Environment

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E-rings (structures shown in Table 1) maintained their ability to inhibit Cdk4 in a selective manner. O NH2

N H

a

N H

O

N H b N H

N H 4

3

5 Cl c + O 6 O N N H 1

Scheme 1. Synthesis of biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-methylamide (1): Reagents and conditions. (a) Ethyl chloroformate, NaOH 4 N, CH2Cl2, 3 h, 90%; (b) LiAlH4, THF, N2, reflux, 1 h, 85%; (c) NaOH 4 N, CH2Cl2, 3 h, 45%. Selective inhibition of Cdk4-cyclin D1. A series of new compounds based on the structure of fascaplysin were identified as specific inhibitors of enzyme Cdk4-cyclin D1 (Table 1). Compound 1 was found to be the most potent inhibitor of Cdk4-cyclin D1 (IC50 = 6 µM) and selective when the Cdk4 IC50 was compared with the IC50s obtained in Cdk2-cyclin A, Cdk1-cyclin B1 and Cdk9-cyclin T1 assays. Compound 1 was better than its other two structural analogs N-(2-(1H-indol-3-yl)ethyl)-4chloro-N-methylbenzamide

(CA223,

7)

and

N-(2-(1H-indol-3-yl)ethyl)-4-tert-butyl-N-

methylbenzamide (CA225, 8),41 with >6-fold better activity against Cdk4-cyclin D1 (Table 1). Unlike fascaplysin, compound 1 does not intercalate with DNA.41 Compound 1 was also tested against 58 represenative kinases at Millipore Bioscience Division, UK. It was found that compound 1 was inactive at the concentration of 10 µM against all kinases including Cdk5-p35, Cdk6-cyclin D1, Cdk7-cyclin H, EGFR, GSK3β, MAPK1, MEK1, PDGFR, Plk3, PKA, PKCα, IGF-1R etc. Results are shown in supporting information. These results support compound 1’s selective ability to inhibit Cdk4-cyclin D1


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enzyme in-vitro while having much reduced or no affinity for other kinases tested. Table 1. Activity of fascaplysin (2) and its non-planar analogs 1, 7 and 8 in in-vitro kinase and DNA binding assays a O

O

Cl C N

Assay

A

D

B N H

O

E A

A

E

B N H

O

N

N

Cl

N E

B N H

A

E

B N H

2

7

1

8

Cdk4-cyclin D1

0.41 ± 0.04

38 ± 6

6.2 ± 0.9

49 ± 6.5

Cdk2-cyclin A

>250

731 ± 26

521 ± 11

658 ± 23

Cdk2-cyclin E

>250

ND

ND

ND

Cdk1-cyclin B1

>250

>500

>500

>500

Cdk9-cyclin T1

>250

>1000

>1000

>1000

5 ± 0.4

Does not displace

Does not displace

Does not displace

EtBr displacement a

IC50 values are presented in µM. All the fascaplysin analogs were dissolved in 100% DMSO and were

further diluted in the kinase assay buffer or the ethidium bromide displacement assay buffer. ND: not determined. In order to understand the observed selectivity towards Cdk4-cyclin D1 versus Cdk2-cyclin A, molecular modeling studies were carried out. The ATP binding sites of these two Cdks are well conserved and share 45% sequence homology with each other, however compound 1 displayed varying degree of affinity to these Cdks. These two Cdks differ from each other, primarily by these three residue sequences: 94-97(Glu-His-Val-Asp)Cdk4/81-84(Glu-Phe-Leu-His)Cdk2, 101-102(Arg-Thr)Cdk4/88-89(LysLys)Cdk2 and Glu144Cdk4/Gln131Cdk2. Compound 1 interacts flexibly with ATP binding pockets of Cdk4 and Cdk2 with 84-fold selectivity towards Cdk4 due to flexible conformational movement of the compound 1 amide bond (1 interacts with Cdk4 and Cdk2 in two different conformational states), which allows free rotation of biphenyl ring. This subsequently leads to loss of major hydrophobic interactions with Cdk2. Two conformations of compound 1 include (a) trans-conformation (green colored ligand in


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Figure 1a; dihedral angle, Ψ = -151.5) which was found to interact selectively with side chains of Arg101 residue of Cdk4 by hydrophobic π-cation interaction. However in Cdk2, this interaction is missing because corresponding Lys88 residue side chain is far away from compound 1 binding cavity; (b) cis-conformation (orange colored ligand in Figure 1a; dihedral angle, Ψ = 2.6) which interacts with Cdk2. On the other hand, the trans-conformation of compound 1 is stabilized in Cdk4 binding site because negatively-charged Glu144 side chain oppose biphenyl aromatic ring. In case of Cdk2, the cisconformation is stabilized because biphenyl aromatic rings are well accommodated by the corresponding neutral Gln131. The amino acid comparison of Cdk4 and Cdk2 binding site residues are shown in Figure 1c.

cis-1 – Cdk2

trans-1 – Cdk4

(a) Protein Cdk4

Cdk2

(b)

Amino acid sequence 94

95

96

97

101

102

144

Glu

His

Val

Asp

Arg

Thr

Glu

81

82

83

84

88

89

131

Glu

Phe

Leu

His

Lys

Lys

Gln

(c) Figure 1. Molecular modeling studies of compound 1 with Cdk4 and Cdk2. (a) Interactions of cis- and trans-conformations of compound 1 with Cdk2 and Cdk4, respectively (cis- and trans- conformations of compound 1 are shown in orange and green colors, respectively. Similarly Cdk2 and Cdk4 residue labels are shown in orange and green colors, respectively). (b) compound 1-Cdk4 interaction histogram ACS Paragon Plus Environment

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with different binding site residues during 10 ns MD simulation of compound 1-Cdk4 protein complex (c) amino acid comparison between Cdk4 and Cdk2 at ATP binding site (black colored residues indicates commonly interacting residues of Cdk4/Cdk2, while red colored residues are those which differ in interaction pattern in these two Cdks). Cancer cell growth inhibition. All the fascaplysin analogues including the most potent compound in the series, compound 1, were tested in a panel of ten different cancer cell lines (some of them are known to be relatively resistant to known chemotherapeutic agents) for their ability to inhibit cancer cell growth in-vitro. The inhibitory effects of compounds were quantified using the MTT assay and IC50s were determined (i.e. the concentration, expressed in µM, of a compound at which 50% cell growth was inhibited). The results of these cell proliferation assays indicated that compound 1 inhibits the growth of cancer cells in-vitro at low micromolar concentrations (Table 2). Amongst all the analogues,41 compound 1 was found to be the most potent molecule even at cellular level. The inhibition of cell growth was both p53 and pRb-independent, the latter indicating that Cdk4-cyclin D1 inhibition is not the only cellular target for the mechanism of action of these molecules.

Table 2. IC50 concentrations expressed in µM for in-vitro cell growth inhibition induced by exposure to compound 1, and its analogues 7-841 for 48 h. O

O

Cl C N

Cell lines

a

A

D

B N H

O

E A

A

E

B N H

O

N

N

Cl

N E

B N H

A

E

B N H

2

7

1

8

LS174T

0.88 ± 0.04

42 ± 2.5

3.5 ± 0.9

18 ± 1

PC-3

0.92 ± 0.06

47 ± 3

6.2 ± 1.1

15 ± 1.5

ND

31± 2.2

4.0 ± 0.3

10.2 ± 0.9

0.69 ± 0.03

27 ± 2.5

3.5 ± 0.6

12 ± 1.8

MiaPaCa A549


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Calu-1

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1.3 ± 0.1

78 ± 3.0

11.5 ± 2.5

52 ± 3.5

NCI-H460

ND

24 ± 1.8

2.0 ± 0.3

9.8 ± 1.0

NCI-H1299

ND

21 ± 0.9

2.5 ± 0.3

11.5 ± 1.6

NCI-H358

ND

26 ± 2.0

2.2 ± 0.6

14 ± 1.4

BNL CL2

ND

29 ± 2.4

2.6 ± 0.9

18 ± 2.0

BNL SV A.8

ND

32 ± 1.0

3.8 ± 0.9

22 ± 1.8

a

LS174T: colorectal carcinoma (p53+, pRb+); PC-3: prostate (p53 null, pRb+); MiaPaCa: pancreatic

(p53His273 mut, pRb+); A549: NSCLC (p53+, pRb+); Calu-1: NSCLC (p53 null, pRb+); NCI-H460: NSCLC (p53+, pRb+); NCI-H1299: NSCLC (p53 null, pRb+); NCI-H358: NSCLC (p53 null, pRb null); BNL CL2: mouse normal hepatic cells; BNL SV A.8: mouse hepatic; SV-40 mediated transformed cells. ND: not determined. Compound 1 retains the G0/G1 block in serum starved p53-null Calu-1 cells. Compound 1, although identified as a Cdk4-specific inhibitor in the in-vitro enzyme assay, generally tends to block cancer cells more profoundly at G2/M than G0/G1 phase of the cell division cycle. Moreover, compound 1 inhibits tubulin polymerization in-vitro with higher potency than it inhibits enzyme Cdk4-cyclin D1. In Calu-1 cells, the mitotic spindle checkpoint is impaired implying that these cells cannot be blocked at G2/M. Hence, it was decided to treat Calu-1 cells with compound 1 after release from cell synchronization at G0/G1 (cells being starved of serum using 0.1% FBS for 24 h). At IC50 and IC70 concentrations of compound 1 the G0/G1 block was either partially or full maintained.40 Since the maintenance of G0/G1 block after serum starvation requires Cdk4 enzyme to be inactive, these results indicate that compound 1 probably inhibits cellular Cdk4 at these concentrations and thereby maintains the G0/G1 block. In the absence of any other kinase inhibitory data, the results obtained with Calu-1 cells tend to confirm the Cdk4 inhibitory potential of compound 1 in live cells.


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Compound 1 at the IC50 concentration induces profound G2/M block in asynchronous cancer cells (p53+) A549 and (p53-null) NCI-H1299 cells. Incubation of A549 (p53+) cells with compound 1 at IC50 concentration for 24 h induces a profound block at G2/M as indicated by the percentage of cells at G2/M. As seen in Figure 2, at the IC50 concentration of compound 1, 89% cells appear to be arrested in the G2/M phase (Figure 2B) and at IC70 concentration, 91% cells blocked at G2/M (not shown). Upon incubation of NCI-H1299 (p53-null) with IC50 concentration of compound 1 resulted in large number of cells (71% cells) accumulation at G2/M (Figure 2D). The results confirm that a profound G2/M block can be observed in cells (A549 and NCI-H1299) where the mitotic spindle checkpoint is intact. Nocodazole and paclitaxel induced G2/M block is maintained by compound 1 in NCI-H358 lung cancer cells. NCI-H358 (p53-null) cells were treated with nocodazole (1 µM, a sub-optimal concentration) only for 18 h in order to induce a partial block at G2/M so that treated cells are minimally stressed. The blocked cells were released in fresh medium when cells readily entered the cell cycle without any apoptosis. When blocked cells were released in the presence of compound 1 for 12 h, cells not only maintained the G2/M block but also >50% of G0/G1 and S phase cells entered G2/M (compare Figure 2E-H). Although only representative histograms of nocodazole treatment are shown, similar results were obtained when paclitaxel blocked cells were released in the presence of compound 1 for 12 h. These observations suggest that, at least, in p53-null/pRb-null NCI-H358 cells, compound 1 maintains the pro-metaphase block induced by nocadozole or paclitaxel during mitosis. Compound 1 blocks NCI-H358 cells in G2/M after release from hydroxyurea-mediated G1/S cell synchronization. Hydroxyurea (250 µM, 18 h) was used to block cells at G1/S (77%; Figure 2J), at a stage of the cell cycle where Cdk2-specific inhibitors normally act. When released in the presence of compound 1, cells proceed from G1/S, confirming that compound 1 does not inhibit cellular Cdk2. Cells ultimately accumulate at G2/M (72%; Figure 2L). These results again indicate that compound 1 have an inherent tendency to induce block at G2/M phase of the cell cycle at least in cells where mitoic spindle checkpoint is normal. ACS Paragon Plus Environment

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Selective killing of SV40 large T-antigen transformed normal mouse embryonic liver cells by compound 1. SV40 large T-antigen inactivates both the tumor suppressor proteins, p53 and pRb, and thereby transforms normal cells into tumorigenic cells. We used normal mouse embryonic hepatic (liver) cells BNL CL2 and its SV-40 large T-antigen transformed counterpart BNL SV A.8 to study the effects of compound 1. The normal cells, upon 48 h incubation with 1, exhibited prominent G2/M arrest at both IC50 and IC70 concentrations with less than 5% cells detected in the sub-G0/G1 phase (Figure 2N) and more than 50% cells appearing at G2/M phase of the cell cycle. Interestingly, in the SV40 large Tantigen transformed cell line, significant apoptotic cell death was observed. After 48 h treatment at the IC50 concentration of compound 1, 31% cells were detected in sub-G1 phase (Figure 2P) indicating apoptosis. The percent apoptosis increased further from 31 to 44% at the IC70 concentration after 48 h incubation (data not shown). Similar results were obtained using normal and SV-40 large T-antigen transformed human lung fibroblast cells, WI-38 (data not shown).

A549

A

NCI-H1299 Control G0 /G1 = 49% S = 29% G2 /M = 22%

PMT4 dna Lin

Compound 1, IC50 , 24 h G 0/G1 = 3% S = 4% G 2/M = 89%

PMT4 dna Lin

B

Control G0 /G1 = 56% S = 21% G 2 /M = 18%

PMT4 dna Lin


C

Compound 1, IC50 , 24 h G 0/G 1 = 22%

D

S = 10% G2 /M = 71%

PMT4 dna Lin

12

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NCI-H358

E

G 0 /G 1 = 59% S = 21% G 2 /M = 18%

Control G0 /G1 = 59% S = 21% G2 /M = 18%

F

G0 /G1 = 22% S = 24% G 2/M = 48%

PMT4 dna Lin

I

Nocodazole, 18 h; Released in medium, 12 h

Nocodazole, 18 h

Control

G0 /G1 = 57% S = 15% G2 /M = 23% Sub G1 = 0%

PMT4 dna Lin

PMT4 dna Lin

Hydroxyurea, 250 µM, 18 h

Hydroxyurea, 250 µM, 18 h Released in compound 1, IC50, 18 h

J

K

G 0 /G1 = 77% S =16% G 2 /M = 6%

G0 /G 1 = 50% S = 12% G2 /M = 33%

PMT4 dna Lin

G 2/M = 72%

PMT4 dna Lin

BNL SV A. 8 (mouse SV40 transformed hepatic cell line)

N

Compound 1, IC50 , 48 h

Control

Compound 1, IC50, 48 h G 0/G 1 = 34% S = 9% G2 /M = 54% Sub G1 = 2%

L

G 0 /G 1 = 16% S = 10%

PMT4 dna Lin

O

G0 /G 1 = 64% S = 23% G2 /M = 13%

P

Sub G 1 = 0%

G0 /G1 = 31% S = 21% G2 /M = 14% Sub G 1 = 31%

Sub G 1

Sub G1 Sub G1

H

G0 /G 1 = 7% S = 10% G 2 /M = 74%

Hydroxyurea, 250 µM, 18 h Released in fresh medium, 18 h

BNL CL2 (mouse normal hepatic cell line)

M

G0 /G1 = 40% S = 10% G2 /M = 42%

PMT4 dna Lin

PMT4 dna Lin

Control

G

Nocodazole, 18 h; Released in compound 1, IC50 , 12 h

Sub G 1

PMT4 dna Lin

PMT4 dna Lin

PMT4 dna Lin

PMT4 dna Lin

Figure 2. Cell cycle analysis of compound 1 treated cells. (A-D) Response of mitotic spindle checkpoint-proficient human lung cancer cell lines, A549 and NCI-H1299 to compound 1. Flow cytometric analysis of asynchronous cells show that majority of cells are arrested in G2/M phase of cell cycle (4n DNA content) in both the cell lines. A549 untreated (A), treatment with IC50 concentration of compound 1 for 24 h (B), NCI-H1299 untreated or control (C) and treatment with IC50 concentration of compound 1 for 24 h (D). (E-L) Analysis of NCI-H358 cells using flow cytometer. The G2/M and G1/S synchronized cells by nocodazole and hydroxyurea respectively were released either in fresh medium or in fresh medium containing IC50 concentration of compound 1 exhibit greater tendency of compound 1 to block the cell growth at G2/M. For nocodazole block experiment, figure show untreated or control cells (E), treated with 1 µM nocodazole for 18 h (F), treated with 1 µM nocodazole for 18 h and released in fresh medium (G) and treated with 1 µM nocodazole for 18 h and released in


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the presence of compound 1, IC50 (H). For hydroxyurea block experiment, figure show untreated or control cells (I), treated with 250 µM hydroxyurea for 18 h (J), treated with 250 µM hydroxyurea for 18 h and released in fresh medium (K) and treated with 250 µM hydroxyurea for 18 h and released in the presence of compound 1, IC50 (L). (M-P) Selective apoptosis in SV-40 transformed cells by compound 1, analysed by FACS. BNL CL2 (mouse embryonic normal hepatic cells) when exposed to compound 1 for 48 h at IC50 concentration (N), show prominent G2/M arrest. BNL SV A. 8 (mouse embryonic SV-40 transformed hepatic cells) underwent apoptotic cell death upon incubation with compound 1. The apoptosis was quantitated by measuring the % cells appeared in SubG1 peak, 31% and 44% cells were found in SubG1 peak after 48 h exposure with compound 1, IC50 (P) concentration. The untreated cells (O) do not show any apoptosis.

Effect of compound 1 on the cellular level of cyclin B1, Cdk1, p53, p21CIP1/WAF1 (p21) and p27KIP1 (p27); analyses in p53+ cells. Western-blot analyses of p53+ cells, A549 and LS174T, after treatment with compound 1 (at the IC50 concentration) for 24 h demonstrated more than 10-fold induction of p53. Thereby, probably the global Cdk inhibitorp21CIP1/WAF1 (p21) was also induced. The levels of p27KIP1 (p27) were also elevated after compound 1 treatment (Figure 3). The proteins Cdk1 and cyclin B1 were down-regulated in the treated cells as compared with untreated control cells. Repression of cyclin B1 and Cdk1 and elevated levels of p21 and p27 is a possible explanation of the G2/M block seen in A549 and LS174T cells that bear functional copies of the tumor supressor protein, p53 (Figure 3). The effects on the cellular level of cyclin B1, Cdk1, p53, p21CIP1/WAF1 (p21) and p27KIP1 (p27); analyses in p53-negative cells were also investigated. Western-blot analyses on proteins from MIAPaCa-2 cells (carrying a p53 mutation) showed that p53, p21 and p27 levels remained unchanged indicating that the p21 and p27 induction seen in p53-positive cells is probably p53 dependent. Interestingly cyclin B1 and Cdk1 levels were elevated and phosphorylation of Cdk1 at the residue Tyr15 remains unaffected (data not shown) indicating that Cdk1-cyclin B1 is still active in p53 mutated cells, after compound 1 treatment.


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Figure 3. Western-blot analysis of p53+ cells, A549 and LS174T. The treatment with compound 1, IC50 concentration for 24 h resulted in induction of tumor suppressor protein p53 and thereby global Cdk inhibitor p21CIP1/WAF1 (p21) was also induced. The levels of global p27KIP1 (p27) were elevated while cyclin B1 and Cdk1 levels were down regulated.

Cell-free tubulin polymerization assays in-vitro indicate inhibition of tubulin polymerization by compound 1. Compound 1 inhibited growth of cancer cells in-vitro at relatively low concentrations than it inhibited the enzyme Cdk4-cyclin D1. FACS analysis and mitotic index experiments (data not shown) had indicated that compound 1 blocked cell growth at the pro-metaphase of the cell cycle. In addition to these observations, the 2-3 fold increase of compound 1 IC50 in cells with impaired mitotic spindle checkpoint suggested another cellular role for compound 1, possibly as a antimicrotubule agent. We investigated the action of compound 1 on tubulin polymerization in-vitro. The results indicated strong antitubulin activity of compound 1 (Figure 4a) while fascaplysin does not show any interaction with tubulin (data not shown). Representative polymerization curves of compound 1, paclitaxel and nocodazole are shown in Figure 4a. Compound 1 inhibits the polymerization of tubulin which is


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concluded from the dose-dependent decrease in Vmax (mOD/min) and reduction of final polymer mass (Figure 4a). When tested at four different concentrations, compound 1 decreased the Vmax from 17 mOD/min to 6.2, 2.1, 1.1 and 0.4 mOD/min at 2.5, 5, 10 and 25 µM respectively. As a consequence of decreased Vmax, upto 80% reduction in final polymer mass has been observed. Microtubules, the key components of cytoskeleton are made up of α/β-tubulin heterodimers. Microtubule assembly has been targeted using number of polymerization inhibitors and inducers, by binding at different sites including a) colchicine binding site; at interphase of the α/β tubulin heterodimer and b) taxol and vinblastine binding site deep inside β-tubulin. The mechanism of tubulin polymerization inhibitor involves binding at the interphase of the α/β tubulin and forming complex with tubulin like colchicine. This complex is added to the microtubule assembly, where it induces unfavorable conformational changes in tubulin dimer (M-loop) and thus further polymerization process gets stopped. Furthermore, tubulin-polymerization inhibitor complex perturbs microtubule growth by sterically blocking further addition of the tubulin dimers to form microtubule assembly.42-44 Upon molecular docking studies performed at colchicine binding site, it was observed that compound 1 binds at α/β-tubulin interphase by H-bonding. Compound 1 interacts with the Thr353 residue of β tubulin by H-bonding. In addition to this, the hydrophobic biphenyl ring fits in hydrophobic core of the β-tubulin formed by Leu248, Ala250, Leu252, Cys241, Leu255, Ala316, Ala317 and Ala354 (Figure 4b). Interestingly, these interactions were missing in fascaplysin. Compound 1 binding at interphase of α/βtubulin, is supposed to induce conformational changes in protein which further perturbs tubulin polymerization to form microtubule assembly.


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control; paclitaxel, 10 µM; Vmax = 19 mOD/min Vmax = 61 mOD/min

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Figure 4. (A) Purified tubulin polymerization assay in vitro. The ability of compound 1 to inhibit tubulin polymerization in vitro was investigated as described in materials and methods. Paclitaxel and nocodazole were used in the assay as known enhancer and inhibitor of tubulin polymerization. Compound 1 was tested for a range of concentrations at which it show inhibition of in vitro cell growth. The change in Vmax value was used as an indicator of tubulin/ligand interactions. The polymerization curves indicate 2.5 µM, 5 µM, 10 µM and 25 µM of compound 1 reduced the Vmax value from 19 mOD/min (control) to 6.2, 2.1, 1.1 and 0.4 mOD/min respectively. The curves shown are average of three independent experiments. (b) Compound 1 interactions at α/β-tubulin interphase.

Compound 1 inhibits paclitaxel-mediated tubulin polymerization and enhances tubulin depolymerization in live cells. The tubulin polymerization experiments performed on whole cells (A549, NSCLC, with intact mitotic spindle checkpoint) confirmed the observation that compound 1 inhibits polymerization of tubulin. Moreover compound 1 also enhances the depolymerization of stabilized tubulin protein. The polymerized and depolymerized (soluble) forms of tubulin were gauged (via Western-blotting) from the accumulation/ disappearence of tubulin protein from pellet and from supernatant fractions of cell lysates treated with IC50 concentration of compound 1. In the first set of experiments where cells were treated with compound 1 in the presence of microtubule stabilizing agent


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paclitaxel, compound 1 showed prevention of tubulin polymerization (mediated by paclitaxel) in a dosedependent manner (Figure 5, upper two panels). In a second set of experiment where intracellular tubulin was stabilized with paclitaxel and then subjected to compound 1 treatment, results show enhancement of tubulin depolymerization with the increasing concentrations of compound 1 (Figure 5, lower two panels).

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Figure 5. Western-blot analysis for effect of compound 1 on tubulin polymerization . Western blots show the response of compound 1 to tubulin polymerization in the presence of paclitaxel and the effect of compound 1 on paclitaxel stabilized tubulin. The assay is performed in whole cells (A549) after 30 min compound treatment at the concentrations indicated in Figure. Supernatant and pellet represent unassembled and assembled tubulin respectively. Tubulin polymerization is detectable by the increase of tubulin in pellet and its disappearance from supernatant. Simultaneous treatment of paclitaxel and compound 1 show inhibition of tubulin polymerization by compound 1 in a dose-dependent manner and resulted in accummulation of unassembled tubulin in supernatant. It was observed that compound 1 also act as an enhancer for tubulin depolymerization in dose-dependent manner when paclitaxel stabilized tubulin was subjected to compound 1 treatment.


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Compound 1 inhibits colony formation efficiency of A549 and Calu-1 cells. The ability of compound 1 in killing cancer cells was explored in the long term survival assay which monitors cancer cells’ colony-forming capacity in-vitro. A549 (with normal mitotic spindle checkpoint normal, pRb+) and Calu-1 (with mitotic spindle checkpoint impaired, pRb+) cells were used in colony formation assays. The colony formation efficiency of A549 and Calu-1 cells is significantly reduced after compound 1 treatment (Figure 6). The concentration at which 50% colony formation efficiency is observed is comaparatively lower than the IC50 concentration for cell growth inhibition in MTT assay indicating that a large number of cells loose the ability to form colonies or do not survive for a long time after compound 1 treatment. For A549 cells, 50% colony formation efficiency was observed at an average 1.4 µM concentration of compound 1 (cell growth inhibition IC50 = 3.5 µM) and for Calu-1 cells 50% colony formation efficiency was observed at an average of 3 µM concentration of compound 1 (cell growth inhibition IC50 = 11.5 µM) (Table 2; Figure 6) indicating that compound 1 may be quite efficacious in in vivo mouse tumor models.


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Figure 6. Compound 1 in colony formation assay. A549 and Calu-1 cells were investigated for their long term survival efficiency after the treatment with different concentrations of compound 1. The colony formation efficiency is expressed as the percentage of colonies formed in the treated cultures compared with untreated cultures. (A) The representative plates show A549 untreated (a), treated with compound 1, 3.12 µM (b), treated with 1, 6.25 µM (c), treated with compound 1, 12.5 µM (d) treated with compound 2, 0.8 µM (e); Calu-1 untreated (f), treated with 1, 3.12 µM (g), treated with compound 1, 6.25 µM (h), treated with compound 1, 12.5 µM (i) treated with compound 2, 1 µM (j). (B) The curves representing colony formation efficiencies of A549 and Calu-1 cells with increasing concentrations of compound 1. All results represent the means and standard deviations of three independent experiments.

Compound 1 induces apoptotic cell death in cancer cells. When A549 cells were treated with increasing concentrations of compound 1 for 24 h, a dose-dependent induction of fragmented nuclei, disrupted cell membrane and apoptotic cell death was observed (Figure 7A-c,d,e). The induction of apoptosis in NCI-H460, NCI-H358 and NCI-H1299 cells was also measured with flow cytometry (Figure 7B). Dose-dependent and time-dependent increase in apoptotic cell death was observed.


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time of incubation Figure 7. Induction of apoptotic cell death analyzed by DAPI staining and FACS. Incubation of A549 cells with increasing concentration of compound 1 for 24 h show the dose dependent induction of fragmented nuclei, distrupted cell membrane and apoptotic cell death (A) The fluorescence microscopic images captured at 40X magnification after staing with DAPI. (A) A549 untreated (a) treated with compound 1, 1 µM (b), treated with compound 1, 2.5 µM (c), treated with compound 1, 5 µM (d), and treated with compound 1, 10 µM (e). The pre-apoptotic and apoptotic cells are indicated with arrows. (B) The percent apoptosis induced in three cancer cell lines by the treatment with compound 1 and determined by FACS analysis. The percent apoptosis is depicted from the percent of cells appeare in Sub G1 peak during cell cycle analysis. The apoptosis show increase with concentration used and time of incubation with compound 1.

Aqueous solubility, cytochrome P450 liability and Caco-2 permeability of compound 1. The solubility of compound 1 in water, phosphate buffer saline (PBS), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was found to be 80, 125, 125 and 5 µg/ml, respectively. In cytochrome P450 liability studies, at 10 µM, compound 1 showed 50, 14, 51 and 19% inhibition of CYP3A4, CYP2D6, CYP2C9 and CYPC19, respectively. In permeability experiment, efflux ratio [Papp (B–A)/Papp


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(A–B)] was 1.1, indicating that compound 1 is not a substrate of efflux pumps. In vivo experiments: Pharmacokinetics and maximum tolerated dose (MTD). In order to check the plasma exposure of the compound in animals, the pharmacokinetics of compound 1 was studied in BALB/c mice following a single 10 mg/kg dose administration by oral route and 1.0 mg/kg dose administration by IV route. The time versus plasma concentration profile of compound 1 is shown in Figure 8. Compound 1 showed good plasma exposure with Cmax of 190 and 371 ng/mL (537 and 1048 nM) by PO and IV routes, respectively. The AUC0-∞ values were 182 and 189 ng·h/mL (514 and 534 nM), respectively.

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IV (1 mg/kg) 0.33 187 189 371 2.52 1.76 88.3 0.5 – 2 h

Oral (10 mg/kg) 1.16 172 182 190 nd nd nd 9.6% 1–4h

B A Figure 8. (A). Time versus plasma concentration profile of compound 1 in BALB/c mice; (B). Pharmacokinetic parameters determined for compound 1 after IV and oral administration.

The studies which allowed determination of MTD-s were performed in Swiss albino mice for two weeks. The concentration at which compound 1 would be tested in vivo was thus ascertained. The loss in animal body weight was considered as a measure of over-toxicity for the test compound. The concentration of the compound at which >10% weight loss was observed was determined and designated as MTD, although usually a weight loss which is below 20% of the initial weight is harmless and animals can recover once the treatment is stopped.45 The toxicity results obtained from these studies indicated that for compound 1 the MTD in mice was ~1000 mpk (mg/kg). Based on the MTD experiment and pharmacokinetics data, compound 1 was tested at 1/10th of MTD concentration i.e. 100 ACS Paragon Plus Environment

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mg/kg in in-vivo xenograft studies. Effects on HCT-116 and NCI-H460 tumor growth inhibition. SCID (Severe Combined Immuno Deficient) mouse, lacking both T and B immune cells, is an established model to study in vivo efficacy of molecules that has potential for the treatment of human cancers. When evaluated, compound 1 showed statistically significant (p 0.25% of Tween 20 in the final formulation of the compound 1. In this study, 6 animals per group were administered with compound 1 at different doses for five days (Q1D x 5) via intraperitoneal route. ACS Paragon Plus Environment

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Animals were monitored for weight loss, morbidity symptoms and mortality for up to two weeks, which was the end of treatment. Significant weight loss was considered when mean animal weight dropped by >10% and was considered highly significant when the drop was >20%. In vivo efficacy study in SCID mice. The in vivo efficacy of compound 1 was determined in two human xenograft models: HCT-116 and NCI-H460 tumor models. HCT-116 experiments: A group of 60 Severely Combined Immune-Deficient (SCID strainCBySmn.CB17-Prkdcscid/J, The Jackson Laboratory, Stock # 001803) male mice weighing 18-25 g and 6-8 weeks old were used for the studies. Human colon carcinoma, HCT-116 cells (ATCC Cat No CCL247) were grown in McCoy’s 5A medium supplemented with 10% FBS (Sigma-Aldrich). The cultured cells were injected subcutaneously into dorsal side of SCID mice at the dose of 6.6 × 106 cells in 0.2 ml of suspension. When the tumor growth reached to about 4-6 mm in diameter (about 5 days), the animals were randomly divided into eight groups, each containing 7 mice. The treatments were continued for 9 consecutive days intraperitoneally. NCI-H460 experiments: A group of 65 Severely Combined Immune-Deficient (SCID strainCBySmn.CB17-Prkdcscid/J, The Jackson Laboratory, Stock # 001803) female mice weighing 15-24 g and 6-8 weeks old were used. Human non-small-cell lung carcinoma, NCI-H460 (ATCC Cat No HTB177) cells were grown in RPMI-1640 medium supplemented with 10% FBS (Sigma-Aldrich). The cultured cells were injected subcutaneously into the dorsal side of SCID mice at a tune of 5.3 × 106 cells in 0.2 ml of suspension. When the tumor growth reached about 4-6 mm in diameter (about 6 days), the animals were randomly divided into eight groups, each containing 6 or 7 mice. The treatments were continued for 9 consecutive days intraperitoneally. Tumor weight measurements: Tumor size was recorded at 2-5 day intervals. Tumor weight (mg) was estimated according to the formula for a prolate ellipsoid: (Length (mm) x (width (mm)2) x 0.5) assuming specific gravity to be one and π to be 3. Tumor growth in compound treated animals was


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calculated as T/C (treated/control) x 100% and growth inhibition percent (% GI) was [100-% T/C]50-52. Body weight measurements: The body weights of animals in different treatment and control groups were monitored by taking the measurements daily during the treatment schedule. By considering the body weight at the start of the treatment as 100%, the percent weight loss was calculated on subsequent days of treatments. Statistical analysis: Data from each experiment was analysed by Microsoft Excel 2000. Statistically significant differences were identified and analyzed using student t-test for multiple comparisons versus control group.50-52 Molecular docking and molecular dynamic simulation. The available crystal structures of Cdk4 are in the apo-form and have several missing residues, thus they cannot be used for molecular modeling.53 In the present study, we used a hybrid homology model of Cdk4 described by Shafiq et al,54 which was developed from the Cdk4-cyclin D apo-crystal structure (PDB: 2W96) by incorporating positions of missing gaps and activation loops from Cdk2 (PDB: 1FIN).55 This hybrid homology model was subjected to protein preparation wizard for H-bond optimization, heterogeneous state generation, protonation and overall minimization. Grid file of docking was constructed using XYZ co-ordinates of the N atom of Val96 residue with a binding site of 12 Å radius grid box (X = −10.521, Y = 208.683, Z = 107.944). For Cdk2 docking, the Cdk2 apo-protein (PDB ID: 1FIN) was subjected to protein preparation wizard for filling missing loops and side chains (using prime), ionization, H-bond optimization, heterogeneous state generation, protonation and overall minimization. Grid file of docking was constructed using XYZ co-ordinates of the N atom of Leu83 residue with a binding site of 12 Å radius grid box (X = − 10.406, Y = 209.105, Z = 107.576). For tubulin docking, the tubulin-colchicine complex (PDB ID: 1SA0) was retrieved from the protein data bank.56 In this complex, protein is heterodimeric in nature, consisting of two α-chains (451 residues), two β-chains (452 residues) and the Stathmin like domain (142 residues). Crystal structure


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was subjected to protein preparation wizard for filling missing loops and side chains (using prime), ionization, H-bond optimization, heterogeneous state generation, protonation and overall minimization. All other ligands, water and ions were removed except colchicine. Grid file for docking was constructed considering colchicine ligand as centroid of grid box of 10 Å size at interphase of α/β tubulin (C and D chains). All ligands were sketched in Maestro, prepared using Ligprep and docked by Glide molecular docking software in XP mode. The Cdk4-compound 1 docked complex obtained from XP-docking was subjected to system builder, in which TIP4P-Ew was used as an aqueous solvent model. The cubic box of 12 Å radius was used to define the core and overall complex was neutralized by adding one Cl- counter ion for simulation. Further this complex was minimized by steepest descent method followed by the Broyden– Fletcher–Goldfarb–Shanno algorithm with convergence threshold of 2.0 Kcal/mol and overall 1000 iterations. MD simulations were carried out at normal temperature and pressure (300 °K and 1.01325 bar, respectively). Thermostat and Barostat method opted was Langevin with ensemble pathway comprising NVT (constant number of particles, volume and temperature) and isotropic coupling method. Overall model system was relaxed before 10 ns simulation and coulombic interactions were defined by short-range cut-off radius of 9.0 Å and by long-range smooth particle mesh Ewald tolerance to 1e-09. AUTHOR INFORMATION. Corresponding Author *Tel: 44(0)116 250 7280; Fax: +44(0) 116 257 7287; E-mail: [email protected] (B.C.) *Tel: +91-191-2569000 (Ext. 345). Fax: +91-191-2569333. E-mail: [email protected] (S.B.B.) *Tel: +91-191-2569111. Fax: +91-191-2569333. E-mail: [email protected] (R.A.V.) Notes The authors declare no competing financial interest.


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IIIM Communication number. IIIM/1731/2014. Author Contributions. SM (Sachin Mahale) performed all Cdk assays and in-vitro biology experiments; PRJ designed fascaplysin analogues for chemical syntheses; BC designed all biology experiments both in vitro and in vivo; SM (Sudhakar Manda) synthesized compound 1 for pharmacokinetic studies; PJ carried out molecular modeling studies; PJ and SBB interpreted modeling results; SSB determined solubility of 1 in various biological fluids; SBB, RAV and BC contributed to manuscript writing. ACKNOWLEDGEMENT. This research work was funded by Cancer Research UK (BC and PRJ) and CSIR 12th FYP BSC-0205 project. ABBREVIATIONS. AUC0-t, the area under the plasma concentration-time curve from 0 to last measurable time point; AUC0∞,

area under the plasma concentration-time curve from time zero to infinity; Cmax, maximum observed

plasma concentration; C0, extrapolated concentration at zero time point; CL, clearance; Cdk4, cyclindependent kinase 4; D/w, distilled water; F, oral bioavailability; mpk, milligrams per kilogram of body weight; MTD, maximum tolerated dose; NSCLC, non-small-cell lung carcinoma; PBS, phosphate buffer saline; rpm, revolutions per minute; SGF, simulated gastric fluid; SIF, simulated intestinal fluid; SCID, Severe Combined Immuno Deficient; t1/2,ß. terminal half life; Tlast, time at which last concentration was found; USP, United States Pharmacopoeia; Vd, volume of distribution; Vdss, volume of distribution at steady state. ASSOCIATED CONTENT. Supporting Information Available. NMR and HPLC Spectra scans and kinase profiling results. This material is available free of charge via the Internet at http://www.pubs.acs.org.


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TOC Graphic

O Cl C N B N H

A

N E

D

A

O Fascaplysin

E

B N H 1

Cdk4-cyclin D1: IC50 = 0.41 µM Cdk2-cyclin A: IC50 = >250 µM EtBr displacement: 5 µM

Cdk4-cyclin D1: IC50 = 6.2 µM Cdk2-cyclin A: IC50 = 521 µM EtBr displacement: Does not displace NCI-H460: IC50 = 2 µM

Anti-tumor activity of compound 1 in SCID mice using Antitumor activity of compound 1 human lung cancer NCI-H460 xenografts

in

NCI-H460 xenograft model

2500

tumor volume, mg

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2000

Control Compound 1, 100 mg/kg

1500 1000 500 0 0

2

4

6

8

10

12

No. of days


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a Nonplanar Analogue of Fascaplysin, Inhibits ... - ACS Publications

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