Novel cellularly active inhibitor regresses DDAH1 induced prostate

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Novel cellularly active inhibitor regresses DDAH1 induced prostate tumor growth by restraining tumor angiogenesis through targeting DDAH1/ADMA/NOS pathway Karthik Reddy Kami Reddy, Chandrashekhar Dasari, Shalini Vandavasi, Sirisha Natani, Bhukya Supriya, Surender Singh Jadav, Sai Ram N, Jerald Mahesh Kumar, and Ramesh Ummanni ACS Comb. Sci., Just Accepted Manuscript • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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ACS Combinatorial Science

Novel cellularly active inhibitor regresses DDAH1 induced prostate tumor growth by restraining tumor angiogenesis through targeting DDAH1/ADMA/NOS pathway Karthik Reddy Kami Reddy,†,‡ Chandrashekhar Dasari,†,‡ Shalini Vandavasi,† Sirisha Natani,†,‡ Bhukya Supriya,† Surender Singh Jadav,† Sai Ram N,# Jerald Mahesh Kumar,# and Ramesh Ummanni†,‡ †

Applied Biology, Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, India

‡

Centre for Academy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, India #

Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, India

Corresponding Author: Dr. Ramesh Ummanni Centre for Chemical Biology, Department of Applied Biology CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Tarnaka, Uppal Road, Hyderabad - 007, India E-mail: [email protected] Phone: 0091-40-27191866

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Abstract

Dimethylarginine dimethylaminohydrolase1 (DDAH1) inhibitors are important therapeutics by virtue of their ability to control nitric oxide (NO) production by elevating asymmetric dimethylarginine (ADMA) levels. In a screening campaign, we identified that DD1E5 (3amino-6-tert-butyl-N-(1,3-thiazol-2-yl)-4-(trifluoromethyl)thieno[2,3-b]pyridine-2carboxamide) inhibits the DDAH1 activity both in vitro and in cultured cells. Mechanistic studies found that DD1E5 is a competitive inhibitor (dissociation constant (Ki) of 2.05 ± 0.15 μM). Enzyme kinetic assays showed time and concentration dependent inhibition of DDAH1 with DD1E5, which shows tight binding with inactivation rate constant of 0.2756±0.015 M-1S-1. Treatment of cancer cells with DDAH1 inhibitors shows inhibition of cell proliferation and a subsequent decrease in NO production with ADMA accumulation. DD1E5 reversed the elevated VEGF, c-Myc, HIF-1α and iNOS levels induced by exogenous DDAH1 overexpression in PCa cells. Moreover, DD1E5 significantly increased intracellular levels of ADMA and reduced NO production suggesting its therapeutic potential for cancers in which DDAH1 is upregulated. In in vitro assays, DD1E5 abrogated the secretion of angiogenic factors (bFGF and IL-8) into conditional media indicating its anti-angiogenic potential. DD1E5 inhibited in vivo growth of xenograft tumors derived from PCa cells with DDAH1 overexpression, by reducing tumor endothelial content represented with low CD31 expression. VEGF, HIF-1α and iNOS expression were reversed in DD1E5 treated tumours compared to respective control tumours. In this work integrating multiple approaches shows DD1E5 is a promising tool for the study of methylarginine-mediated NO control and a potential therapeutic lead compound against pathological conditions with elevated NO production such as cancers and other diseases.

Key words: Dimethylarginine dimethylaminohydrolase-1, Asymmetric Dimethylarginine, Tumor growth inhibition and Angiogenesis

Abbreviations:

DDAH1:

Dimethylarginine

dimethylaminohydrolase-1,

Asymmetric Dimethylarginine, NO: Nitric Oxide, PCa: Prostate cancer


ADMA:

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Introduction Prostate cancer (PCa) is second leading cause of cancer related deaths in men and currently early diagnosis and treatment remain key approaches for patient care. High rate of mortality in PCa patients is mainly due to metastasis

1-2

. PCa metastasis is dependent upon

the aggressiveness of tumor and its surrounding blood and lymph vessels 2. In PCa, tumor progression and angiogenesis are positively regulated by neovascularisation. Oncogenic activation of PCa progression and metastasis involves many regulatory factors. But it is evident that nitric oxide (NO) is one of the regulators that maintain and promote (neovascularisation) angiogenesis 3. Apparently NO plays important roles in various stages of carcinogenesis such as DNA damage, oncogene activation, inhibition of DNA repair enzymes and tumor suppressor genes, and the modulation of apoptosis and metastasis 4. Significant NO production has been found in malignant human breast, neuronal, gastric, cervical, prostate and ovarian cancers, but not in the surrounding benign tissues. NO produced by tumor cells helps the tumor growth by enhancing vascular permeability, extracellular matrix degradation, endothelial cell proliferation, migration and stimulates the expression of vascular endothelial growth factor (VEGF)

3, 5

. In neuronal, breast, gynaecological, head,

prostate and neck tumors, NO levels have been shown to positively correlate with increasing tumor grade 6-7. Although the detailed mechanism of NO participation in tumor biology is still being elucidated, there is increasing evidence that its biosynthesis plays an important role in angiogenesis and tumor progression and thus the inhibition of NO production has been suggested as a possible antitumor therapeutics 6. Since nitric oxide synthase (NOS) inhibitors block the physiological production of NO by immune cells, an alternative target has been suggested for blocking NO production in tissue selective manner. One such regulatory mechanism involves pools of endogenously produced NOS inhibitors, Nω-methyl-Larginine (NMMA) and asymmetric Nω, Nω-dimethyl-L-arginine (ADMA) which inhibits all isoforms of NOS 8. The concentrations of these methylated arginines are controlled in turn by another enzyme, dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes both the substrates thus relieving the inhibition of NOS and promoting NO biosynthesis 9. DDAH is a cysteine hydrolase enzyme that is expressed in all nucleated mammalian cells in two isoforms (DDAH1 and DDAH2). About 80% of endogenous ADMA is metabolized by DDAH1 10. Indirect regulation of NO production through DDAH1 inhibition does not effect NO production in immune cells without compromising their ability to fight



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infections. However, since DDAH1 is not much expressed in immune cells especially in macrophages and also in endothelial cells, it does not regulate vascular NO production which predominantly is regulated by DDAH2

11-13

. Therefore, the development of potent and

selective DDAH-1 inhibitors may enable the tissue selective inhibition of NO production. Notably, overexpression and elevated DDAH1 activity have been detected in hepatocellular carcinoma, glioblastoma, melanoma, breast and prostate tumors

14-18

. DDAH1 expression is

correlated with aggressiveness of the PCa and Glioma and regulates angiogenesis by altering the VEGF cascade 15, 19. Recent studies manifest that targeting DDAH1 may be utilized to accumulate ADMA in conditions characterized by excessive NO production to inhibit NOS activity. To date, only a limited number of human DDAH1 inhibitors have been identified, and many of these are substrate analogues. A few classes of substrate-dissimilar inhibitors have been reported, including 2-chloroacetamide, indolylthiobarbituric acids, pentafluorophenyl sulfonates, and 4-halopyridines. Most of these have been derived from studies using the Pseudomonas aeruginosa isoform of DDAH, which has only 25 % sequence identity to human DDAH1

11

.

Only few inhibitors like Ebselen, 4-hydroxy-2-nonenal (4-HNE), PD404182 and proton H+/K+ ATPase pump inhibitor (PPIs) are identified against human DDAH1 protein

20

.

However, none of these selective DDAH1 compounds were studied further. Hence, the attractive target rationale combined with previous success in identifying DDAH1 role in PCa progression prompted us to identify potential human DDAH1 inhibitors capable of inhibiting PCa progression and also useful as chemical probes to investigate DDAH1 role in pathophysiology of cancers.

Results and Discussion: Identification of DDAH1 small-molecule inhibitor: Identification of novel molecules/targets involved in the cancer progression is an important area in molecular targeted cancer drug discovery and development

21

. The role of

DDAH1 expression and /or activity in the PCa and its importance in PCa progression through regulating angiogenesis is well studied

19

. Disruption of angiogenesis through

DDAH1/ADMA pathway with novel molecules is an emerging area of clinical investigation in multiple cancer types, including PCa 20. This alternative target to inhibit NO production in cancers is increasingly recognised

3, 14-15, 18-19, 22

. To identify small molecule DDAH1

inhibitors, we have adapted fluorescence assay reported by Stone EM & Fast W 23 with minor modifications. We constructed a recombinant vector (pET28a(+)-hDDAH1) expressing His


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tagged human DDAH1 in E. coli. The His tagged protein was purified in active form and showed a single band around 37 kDa on SDS-PAGE. Activity of the recombinant human DDAH1 was assessed by using an alternative substrate S-methyl-L-thiocitrulline (SMTC) (Supporting figure 01 and Supporting table 03) and this fluorescent SMTC assay is optimized to 96 well plate format. We conducted semi HTS of approximately 5500 compounds from our internal chemical library store at National MolBank facility, CSIR-IICT, India. From primary screening results for total 5500 compound library, composite z-scores were calculated using percent enzymatic inhibition and standard deviation from triplicate experiments. The z score was calculated using equation 1. 𝑧 𝑠𝑐𝑜𝑟𝑒 =

% 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛−𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑀𝑒𝑎𝑛 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐷𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛

Eq: 1 24

The cut-off mean z-score of more than 2 is set to select compounds showing 60% inhibition in primary assay (Figure 1A). From the following orthogonal secondary assay of 59 compounds (Z score > 2), we identified 10 compounds with a half-maximal inhibitory concentrations (IC50) value (< 15 μM) against DDAH1 in the micro molar range (Figure 1B and 1C). Further, we evaluated whether selected compounds will elicit desired cellular phenotype or off-target effects when used at appropriate concentrations. In cell based DDAH1 activity assay, three out of ten compounds showed cellularly active enzyme inhibition (Figure 1D). This may be due to the reason that the remaining hits may not be working at cellular level against DDAH1 enzyme activity. From the above results we finally selected three compounds i.e., DD1E5 (IC50 = 2.1±0.21 μM), DD1C7 (IC50 = 3±0.21 μM) and DD1G7 (IC50 = 12.7±3 μM) for further studies. Many of DDAH1 inhibitors reported till date

are structurally similar to its substrate (ADMA) except for a few inhibitors25.

Interestingly, identified DDAH1 inhibitors from the present study are structurally diverse to enzyme substrates (Figure 1E, 1F and 1G). The advantage of structurally diverse inhibitors from structurally similar inhibitors is non-requirement to compete for Y+ transporters and unintended inhibition of arginase leading to the counterproductive increase in the NO biosynthesis25. To understand the specificity of the selected compounds against DDAH1 we searched for the structural analogues for the selected three compounds from 5500 compound library by using near neighbour search with 0.7 tanimoto cut off score. Top analogues (DD1E5: 11 analogues, DD1C7: 06 analogues, DD1G7: 11 analogues) from the tanimoto cut off score for the three compounds did not pass the primary screening. Further, we conducted a substrate competition assay to analyse the mode of action. Characterization of the DDAH1 inhibition



by the selected compounds at various concentrations shows that the double reciprocal lines are intersecting at 1/Vmax in lineweaver-burk plot (Figure 2A, 2B and 2C). Addition of these compounds has a profound effect on apparent KM value, but not on the Kcat value (Figure insets 2A, 2B, 2C and Supporting table 04). The apparent KM is increased with the increased concentration of compounds whereas the Kcat value remains constant at all concentrations of test compounds.

Figure 01: A. Screening of small molecule library representing Z-score values against the purified human DDAH1 enzyme. Z-score cut off >2 identified 59 potential compounds against DDAH1 in primary screening. B. From primary hits, 10 compounds showing >75% inhibition in secondary orthogonal assay (colorimetric) are selected. C. IC50 values of 10 selected compounds against human DDAH1 protein shows only 5 compounds inhibited DDAH1 with IC50 < 15µM. D. Inhibitory potential of identified hits against cellular DDAH1 activity. In cell based citrulline assay cells exposed to 10 hit compounds, 3 hits significantly inhibited DDAH1 activity. Homocysteine (2 mM) was used as positive control. E, F, G. Molecular structures of selected compounds DD1E5, DD1C7 and DD1G7. Experiments are performed twice repeatedly with triplicates and results are mentioned as ± SD with statistical significance * = p ≤ 0.05, **= p ≤ 0.01, *** = p ≤ 0.001.


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This observation indicates that three compounds are showing competitive inhibition model with Ki values of 2.05±0.15 μM, 2.7±0.4 μM and 4.9±0.2 μM, respectively. Evaluation of relative affinity of lead molecules to the target enzyme may determine the dissociation constant for inhibition (Ki). Determining the inhibition modality and dissociation constants (Ki) of selected compounds under different conditions provides possible binding confirmations between enzyme and compounds 21, 26, this was supported by the computational molecular modelling studies on DDAH1 interactions with the selected 3 compounds. Understanding the structure of the protein-ligand interaction is important to determine molecular mechanism of enzyme inhibition. Herein, we used computational methods to probe possible protein-ligand interactions. As a reference, the N5-(1-iminopropyl)-L-ornithine binding site located in chain A of DDAH1 was employed for current docking experiment. A few vital amino acid residues of DDAH1 important for allowing the reference ligand binding include Cys274, His173, Arg145, Leu30, Asp73, and Asp269. The Arg145 in the active site of both wild type and its mutant (inactive enzyme) forms the hydrogen bonds with carboxylic acid of N5-(1-iminopropyl)-L-ornithine

27

. The thieno ring of trifluoromethyl-thieno[2,3-

b]pyridine of compound DD1E5 exhibited π-cation interaction with Arg145. The N-thiazolyl ring displayed the π- π cation interaction with His173 located deep in the cavity of binding site (Figure 2D). In case of compound DD1C7, the thioxothiazolidinylidene portion and phenyl group present on nicotinonitrile ring were found to be having significant interaction with Arg145. The formation of salt bridge with free NH of thiazolidinylidene was noticed and nicotinophenyl group showed π-cation interaction with Arg145. The orientation of nicotinonitrile ring was found to be fitted inside the active site (Figure 2E). Similarly, the dihydroquinolin ring of azaflavone (DD1G7) displayed π-cation interaction with Arg145. The dimethoxy phenyl ring was found to be establishing π-π cation interaction with His173 and the methoxy group exhibited the hydrogen bond with Ser176 (Figure 2F). By visual inspection from the above docking data it has been concluded that, all of the selected three compounds were accommodated in the N5-(1-iminopropyl)-L-ornithine binding site and interaction with Arg145 might play a significant role for their DDAH-1 inhibition. Time dependent inactivation of DDAH1 at different concentrations of selected compounds is fitted in equation 2 (Figure 3A, 3B and 3C). The concentration response relationships reflect the ideal behaviour of inhibitors due to the stoichiometric binding to the enzyme 26.



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Figure 02: Lineweaver-burk plots of selected DDAH1 inhibitors. DDAH1 activity was assessed by adding different concentration of DDAH1 inhibitors in the assay mixture of 0 (●), 1 (○), 2.5 (▼), 5 (▲) and 10 (■) µM, and using different concentrations of ADMA as substrate at pH 7.5, 37 0C. The double reciprocal plots (DD1E5 (A), DD1C7 (B) and DD1G7 (C)) representing here for the easy interpretation of data, intersects at 1/V max with insets showing variation in the apparent KM with compound concentrations. All experiments are performed twice repeatedly, each with triplicates and error bars are mentioned as ±SD. Modelled structures of selected 3 DDAH1 inhibitors DD1E5 (D), DD1C7 (E) and DD1G7 (F) bound to the DDAH1 active site pocket. Each panel shown from a similar vantage point and shows a cut away view of the active site cavity’s surface. The enzyme backbone is shown in ribbons with selected residues depicted in stick form and ligands are shown in stick form.

Secondary plots of these reaction rates against different concentration of selected compounds gave the linear plot except for DD1C7 (Figure 3E) showing time and concentration dependent inhibition of DDAH1, yielding a second order rate constant (DD1E5: 0.2756±0.015 M-1S-1, DD1G7: 0.089±0.004 M-1S-1) (Figure 3D and 3F).

To

understand the resident time of the compounds on its target enzyme, second order rate constant (Kobs) was measured. From the mechanistic studies we observed that selected compounds were displaying slow off rates which may bind tightly or covalently to the DDAH1 protein. Further, the covalent docking studies employing Arg145 and Cys274 residues as site for reaction has been performed. The compounds, DD1E5 and DD1C7


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formed covalent bond with the guanidine moiety of Arg145 and the plausible mechanism behind this might be nucleophilic substitution. The nucleophilic addition type of covalent bond of DD1G7 with Cys274 was noticed (Figure 3G, 3H and 3I). To better understand reversibility/ irreversibility of selected compounds we performed the separation method using dialysis. DDAH1 inactivated with the selected compounds was dialysed overnight and assayed for recovery of enzyme activity. The DDAH1 activity in presence of compounds after dialysis does not recovered when compared to the non-dialysed samples (Figure 3J). This implies that the nucleophile in the enzyme active site and week electrophiles sites of the compounds may be forming a covalent adducts with DDAH1 showing irreversible inhibition. This was supported by the computational molecular modelling studies on covalent bond formation with DDAH1 and the selected 3 compounds. Compounds that display slow off rates from their targets may offer important advantages in clinical medicine. From our studies, DD1E5 shows significant time and concentration dependent inhibition of DDAH1 compared with other two compounds. This observation emphasizes that DD1E5 is superior to the remaining two compounds for the effective inhibition of cellularly active DDAH1. During lead optimization it is important to understand the cellular phenotype in the presence of selected compounds

26

. From the results obtained,

DD1E5 demonstrated more inhibitory potential against DDAH1 than DD1C7 and DD1G7. Thus, DD1E5 is more effective competitive and tight binding DDAH1 inhibitor. A.

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Figure 03: Time and concentration dependent inhibition of human DDAH1 enzyme with selected compounds shows a time dependent loss in DDAH1 activity is observed after pre incubation with 5 (●), 10 (○), 20 (▼), 40 (Δ) and 80 (■) µM DD1E5 (A & D) and DD1G7 (C & F) with observed inactivation rates of 4.85, 11.1, 12.85, 14.55, 29.65 s-1, and 2.6, 2, 2.8, 2.7, 8.8 s-1, respectively. Observed pseudo first-order inactivation rates vary linearly with DD1E5 and DD1G7 concentration and are fit using a second order rate constant of 0.2756±0.015 M-1S-1 and 0.089±0.004 M-1S-1, respectively. We didn’t observe time dependent inactivation of DDAH1 with DD1C7 compound (B & E). Computational method used to determine the predictive covalent bond formation with the selected three compounds. DD1E5 (G) and DD1C7 (H) forming covalent bond (arrows) with Arg145 residue of active site with nucleophilic substitution and nucleophilic addition of covalent bond of DD1G7 (I) with Cys274 (arrow) is noticed. J. DDAH1 inactivated by the selected three compounds (DD1E5, DD1C7 and DD1G7) were dialysed overnight and tested for the recovery of enzyme activity. All experiments are performed two times in triplicates with error bars mentioned as ±SD.

Inhibition of cell proliferation by DDAH1 inhibitors Because DDAH1 promotes growth and survival of different cancer cells

8, 15

, first we

examined the effect of DDAH1 inhibitors in LNCaP and PC3 cells in which DDAH1 expression is elevated compared to normal prostate cells (WPMY-1 non-cancerous cells) (Supporting Figure: 02A).


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Figure 04: Characterization of selected DDAH1 inhibitors in PCa cell lines with overexpression of DDAH1. A. RNA and protein level overexpression of DDAH1 in LNCaP and PC3 cells stably transduced for enzyme overexpression. Values in parentheses indicate relative fold difference compared with control determined by densitometric analysis. B & C. DDAH1 inhibitors attenuate proliferation of LNCaP and PC3 cells induced by elevated expression and activity of DDAH1. D DDAH1 activity is elevated in LNCaP and PC3 cells due to stable overexpression of DDAH1 exogenously. DDAH1 inhibitors diminished the elevated enzyme activity in both PCa cells lines. E & F. Due to elevated enzyme activity, ADMA levels are depleted whereas NO levels increased in DDAH1 positive PCa cells. DDAH1 inhibitors restored the cellular ADMA levels there by inhibited NO production in PCa cells with elevated DDAH1 activity. All experiments are in biological triplicate with ±SD, statistical significance * = p ≤ 0.05, ** = p ≤ 0.01.



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Cytotoxic evaluation of the selected three compounds against PCa cells (androgen independent PC3 & androgen dependent LNCaP) shows significant inhibition on cell growth with IC50 values below 15 μM concentration (Supporting Table: 05). To systematically characterize the identified inhibitors, we developed PCa cells with increased DDAH1 specific activity by stably overexpressing enzyme through retroviral mediated gene transfer (Figure 4A). Treatment with 10 μM of test compounds attenuated the enhanced proliferation rate by DDAH1 in both the PCa cells. The most potent inhibitor DD1E5 (in target-based assay) is more effective in inhibiting cell growth (Figure 4B and 4C). All the three compounds inhibited elevated DDAH1activity in PC3 and LNCaP cells (Figure 4D) confirming their specificity against DDAH1. Enzyme substrate ADMA is accumulated in cells treated with compounds compared to mock treated DDAH1 positive cells, supported the above observation (Figure 4E). These 3 test compounds are also able to inhibit endogenous DDAH1 activity and NO production in LNCaP and PC3 cells (Supporting Figure: 02B & C). In PCa cells, DDAH1 expression regulates the NO synthesis through ADMA metabolism 3. Upregulation of DDAH1 led to reduced ADMA and elevated NO levels in cancer cells

28

. Since ADMA is a direct inhibitor of NOS enzymes, the activity of the

compounds eventually regulates cellular NO levels. In a recent study, Efthymia et al., reported the role of DDAH1 in progression of glioblastomas highlighting the fact that dependency of DDAH1 on NO is essential to regulate the tumor growth and vasculature

29

.

Accordingly, in our study elevated NO produced due to the enzyme activity in DDAH1 positive cells is inhibited by DDAH1 inhibitors (Figure 4F and Supporting Figure 03), indicating on-target effect of identified inhibitors. Confirming this cellular effect on proliferation is due to the DDAH1 inhibition, DDAH1 knockdown using CRISPR-Cas9 mechanism in both PC3 and LNCaP cells led to decreased rate of cell proliferation compared to cells with normal DDAH1 expression (Supporting Figure 04A and B).

Inhibition of tumor angiogenesis by DDAH1 inhibitors Pathological NO plays a crucial role in angiogenesis of PCa via regulation of several pro angiogenesis factors expression. Hence, the identified compounds through DDAH1 inhibition may regulate tumor angiogenesis negatively and further inhibit tumor progression. To evaluate the effect of DDAH1 inhibitors on angiogenesis, we measured the expression of pro-angiogenic factor VEGF in DDAH1 positive cells treated with compounds. As expected VEGF should be down regulated, these compounds blocked expression of VEGF compared to control cells with DDAH1 overexpression in which VEGF levels are elevated. Meanwhile,


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other NO regulating genes such as iNOS, c-Myc and HIF-1α are also downregulated indicating that the DDAH1 inhibitors attenuate angiogenesis potential of DDAH1 positive cells (Figure 5A). Moreover, NO contributes to the angiogenesis in cancer cells through secreting the angiogenic factors to its surroundings to stimulate the endothelial cells 5. Interestingly, in PCa cells exposed to test compounds DDAH1 expression is marginally decreased compared to mock treated cells. This may be due to the fact that DDAH1 inhibition disrupting DDAH/ADMA cycle might be controlling negative feedback mechanism by accumulated ADMA. Vascularization of tumor is tightly regulated by angiogenic factors secreted by malignant cells through different regulatory pathways30. In vitro tube formation assay by endothelial cells is the best suitable assay to identify angiogenesis inhibitors31. When conditioned medium (CM) from both DDAH1 positive LNCaP and PC3 cells, treated with either compounds or vehicle was added to the HAEC cells independently, the tube formation ability of these cells was affected. The CM collected from PCa cells treated with test compounds significantly inhibited formation of elongated capillary like structures compared to conditioned media from vehicle treated control cells (Supporting figure 03B). This may be due to the response to angiogenic signals present in conditioned media. Sunitinib (100 M) and PMA (0.25µM) were used as respective controls for the experiment. The quantitative data measuring mean % branching confirmed significant effect of DDAH1 on promotion of angiogenesis by releasing angiogenic factors in to CM (Figure 5B). The branching pattern shows the 50% reduction of branching when compared to the vehicle control confirming significant effect of inhibitors on DDAH1 and further prohibit angiogenesis factors release in to CM to arrest angiogenesis. DDAH1 promotes secretion of proangiogenic factors such as basic fibroblast growth factor (bFGF) and IL8 by PCa cells in vitro19. The angiogenic switch is induced when factors like VEGF, PDGF, bFGF, EGF and IL8 favours the microenvironment surrounding the tumor32. Concurrently, DDAH1 inhibitors restrain release of bFGF and IL8 in both DDAH1 overexpressing PCa cells (Figure 5C & D, p ≤ 0.01). Therefore, this result indicates that DDAH1 inhibitors cause accumulation of ADMA hindering NO production, which in turn decreases pro-angiogenic signals arresting angiogenesis.



Figure 05: Inhibition of DDAH1 activity modulates angiogenic pathway in PCa cells. A. DDAH1 activity regulates NO regulatory genes through ADMA metabolism. Increased DDAH1 activity led to upregulation of VEGF, iNOS, c-Myc and HIF-1α expression in PCa cells where as DDAH1 inhibitors reversed the positive regulation of pro angiogenic factors. Values in parentheses indicate relative fold difference compared with control determined by densitometric analysis. B. In vitro angiogenesis assay by measuring tube formation ability of HAEC in conditioned medium collected from LNCaP and PC3 cells with overexpression and inhibition of altered DDAH1. The proliferation rate of HAEC forming tube like network is positively correlated with DDAH1 expression. DDAH1 inhibitors decreased the amplitude of angiogenic signals there by attenuated HAEC cells forming tube like network. C & D. Elevated angiogenic factors like bFGF and IL8 in conditioned medium due to DDAH1 overexpression are restored and depleted by DDAH1 inhibitors in both PCa cells. Data presented are in biological triplicate with ±SD, statistical significance * = p ≤ 0.05, ** = p ≤ 0.01.


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DDAH1 inhibitors arrest migration and invasion of PCa cells. Enhanced tumor growth and angiogenesis lead the cancer cells to migrate into surrounding tissues, which is one of the hallmarks of cancers.

Particularly, when

proangiogenic factors activate their specific receptors, tumor cells release proteases to degrade the basement membrane for cell migration 1. Angiogenesis in cancers is required for expansion of primary tumor mass and new blood vessels formation is the entry point of the cancer cells into circulation

32

. We recently reported that DDAH1 positively regulated PCa

cells migration and invasion in in vitro 19.

Figure 06: DDAH1 inhibitors arrest migration and invasion of PCa cells induced by elevated enzyme activity due to its exogenous overexpression. A. Selected DDAH1 inhibitors ceased the migratory and invasive potential of LNCaP and PC3 cells stably overexpressing DDAH1 (Arrows show migrated & invaded



Page 16 of 30

cells). B & C. Quantitative measurement of % cells migrated and invaded in presence and absence of selected enzyme inhibitors. Data presented are the mean three individual experiments with ±SD, statistical significance ** = p ≤ 0.01, *** = p ≤ 0.001. D. DDAH1 overexpression activated the phosphorylation of FAK (Y397) that is involved in migration and invasion of PC3 and LNCaP cells. DDAH1 inhibitors significantly reduced the activation of FAK (phosphorylation at Y397) in DDAH1 overexpressed PCa cells there by discontinuing the migration and invasion induced by DDAH1. Values in parentheses indicate relative fold difference compared with respective controls determined by densitometric analysis. Only representative blots were presented here.

In this study also, as compounds inhibited DDAH1 induced release of angiogenic factors we tested their effect on cell migration and invasion using boyden chambers. DDAH1 overexpression in PC3 and LNCaP cells enhance the metastatic potential by 2-3 fold compared to control cells. DDAH1 inhibitors reversed its effect on migration and invasion of both cells with elevated DDAH1 activity (Figure 6A, 6B and 6C). Mechanistic studies revealed that DDAH1 regulate cell migration and invasion through NO induced tyrosine phosphorylation of FAK

19, 33

. DDAH1 enhances the metastatic potential of PCa cells by

activating the FAK and SRC kinases through phosphorylation

19

. Since FAK mediates the

invasive motility of tumor cells, the disruption of FAK activity reduces the invasiveness of tumor cells

34

. We examined the effect of DDAH1 inhibitors on phosphorylation of FAK.

Concurrently, DD1E5, C7 and G7 inhibited phosphorylation of FAK at Y397 site by inhibiting DDAH1 activity in both PCa cells examined (Figure 6D). Activated FAK phosphorylates many FAK substrates such as Crk associated substrate (CAS), Paxillin, and p190RhoGAP playing a central role in cell migration

35

. The results obtained in the present

study confirm that DDAH1 inhibitors are able to cease cancer cells from migration and invasion into the neighbouring tissues and organs.

DDAH1 inhibitor suppresses PCa tumor growth. We observed that DD1E5 binds to the active site of DDAH1 and inhibits enzyme activity in vitro and as a result inhibits proliferation of PCa cells and regulates NO production via elevating ADMA levels. From the three identified compounds DD1E5 is found to be more effective in all in vitro and in silico studies compared to DD1C7 and DD1G7. Very recently, we revealed that DDAH1 overexpression promotes PCa tumor growth in vivo. We reported distinct angiogenesis model with stable overexpression of DDAH1 in PCa cells

19

.

Because DDAH1 promotes PCa tumor growth, we examined the effect of DD1E5 on inhibiting DDAH1 positive tumor growth in vivo. The xenograft tumors generated with PC3 cells overexpressing DDAH1 showed significant increase in growth (450.29 ± 76.16 mm 3;


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463 ± 52.1 mg) compared to pMSCV-Empty xenograft (267.16 ± 69.8 mm3; 265.55 ± 20.8 mg). The mock treated xenograft (474.27 ± 58 mm3; 479.2 ± 51.3 mg) also showed similar growth rate as pMSCV-DDAH1. The xenograft mice received DD1E5 (50mg/kg) compound for 3 weeks (twice a week) though IP route showed significant decrease in both tumor growth (328.66 ± 80.24 mm3; 281.8 ± 12.4 mg) as well as tumor volume and the same is observed with pMSCV-Empty xenograft. Interestingly, in mice administered with DD1E5 the tumor growth is similar to untreated and mock treated xenografts up to third treatment, and after third treatment growth is delayed until the last treatment compared to controls. Since DDAH1 is an enzyme, its enzymatic activity degrading ADMA promotes PCa cells growth or protein itself may also contribute to tumor growth. To better understand this, we created DDAH1 negative PC3 cells using DDAH1 specific CRISPR-Cas9. Extending this observation, in vitro proliferation of PCa cells (PC3 and LNCaP) with DDAH1 knockout showed significant decrease in proliferation compared to control cells (Supporting figure 04). The DDAH1 negative cells showed significantly delayed growth (145.25 ± 35.39 mm3; 199.2 ± 11.4 mg) compared to pMSCV-Empty xenograft (Figure 7A, 7B, 7C). The treatment of DD1E5 compound to the xenograft bearing nude mice did not result in significant loss or gain in body weights, signs of toxicity or any mortality compared to the respective control animals (Figure 7D). With this observation, it is clear that DDAH1 inhibition effects in vivo tumor growth and DDAH1 is one of the genes essential for the tumor growth in PCa.



Figure 07: Inhibition of DDAH1 hydrolase activity delays the tumor growth in vivo. A. The representative images of male nude mice bearing tumors from PC3 cells engineered for stable overexpression of DDAH1 or control cells with empty vector. In the mice bearing tumors with DDAH1 positive cells treated with DD1E5, tumor growth was inhibited significantly compared to untreated or vehicle treated mice. DDAH1 knock out by CRISPR- Cas9 mechanism in PC3 almost disposed PC3 cells to grow as tumor in mice. B & C. Graphs representing in vivo tumor growth rate and tumor weights of xenograft tissues, respectively. D. Treatment of DD1E5 compound to the xenograft bearing mice did not result any significant loss or gain of body weight compared to the respective controls. Data presented are the mean of 5 mice from each group with ±SD, statistical significance * = p ≤ 0.05, *** = p ≤ 0.001.

All xenograft tumors were collected and subjected to histological evaluation to confirm effect of test compound on growth and histology of the tumors. In histopathology examinations with H&E staining, all tumors from each group grew in a nodular shape and cells from all xenografts are restricted to sub cutaneous region. To measure the proliferative index of the xenograft tumors we counted the mitotic figures appeared in a microscopic field and mitotic index was calculated by counting the mitotic figures in at least 5 microscopic fields. Average mitotic figures appeared are significantly high in the pMSCV-DDAH1 untreated and pMSCV-DDAH1 mock treatment xenograft sections compared to pMSCVEmpty xenograft tumors (>2 fold increase, p value ≤ 0.001). Upon DD1E5 treatment, nuclear regions are very condensed in pMSCV-DDAH1 treatment xenografts compared to the mock and untreated xenografts. In DD1E5 treated xenograft tumors tissues significant necrosis was observed. Moreover, in CRISPR-Cas9 DDAH1 xenograft sections very few mitotic figures appeared compared to the pMSCV-DDAH1 (>2 fold decrease, p value ≤ 0.001, Figure 8A and 8B).


Page 18 of 30

Page 19 of 30

A.

pMSCV-DDAH1

pMSCV-DDAH1 Mock

pMSCV-DDAH1 DD1E5

CRISPR Cas9 DDAH1

H & E Staining

pMSCV-Empty

Bar : 40µm

B.

***

Figure 08: Histology evaluation of xenograft tumors A. H &

8

and necrosis indicated by arrows. B. Analysis of mitotic

6

index in xenograft tumors sections. Results from quantitative 4

cell count analysis of mitotic cells are shown as mitotic

2

0

***

VEm pt SC y VD D pM A H SC 1 Untreated VD D pM A H SC 1 Mock treated VD D A C H R 1 DD1E5 IS P D R D -C A H AS 1 9

***

index. Data presented are the mean of 5 mice from each group with ±SD, statistical significance *** = p ≤ 0.001.

pM

Mitotic index (%)

E staining of representative tumor xenograft showing mitosis

pM SC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To confirm the DDAH1 overexpression and knockout in PC3 cells injected in nude mice, immunohistochemical analysis was performed. Immunostaining of DDAH1 in xenograft sections confirmed the overexpression in pMSCV-DDAH1 cells and knockout in CRISPR-Cas9 DDAH1 PC3 cells (Figure 9A). Further, immunohistochemical analysis was performed to verify DD1E5 role in expression of pro angiogenesis and growth factors. Very weak immunostaining for VEGF, HIF-1α and NOS on DDAH1 positive PCa xenograft sections treated with compound confirmed that DD1E5 inhibited active DDAH1 in tumor cells as expected. Very strong staining is observed in pMSCV-DDAH1 untreated and pMSCV-DDAH1 mock treated cells compared pMSCV-Empty control cells. Corroborating these results that inhibition of DDAH1 enzyme activity reduced tumor growth, we found weak immunostaining for VEGF, HIF-1α and NOS with no DDAH1 expression in xenograft sections with DDAH1 negative PC3 cells (Figure 9A). Taken together, it is clear that inhibition of DDAH1 with DD1E5 in DDAH1 positive xenografts show delayed growth and decreased expression of pro-angiogenic factors. Growth of the PCa tumor is mainly based on the blood vessels (Micro vessel density, MVD) surrounding the tumor. MVD is significantly higher in the metastatic prostate tumors compared to the localized tumor 36. To investigate efficacy of DD1E5 against the angiogenic



Page 20 of 30

potential mediated by DDAH1, we stained CD31 endothelial marker to measure MVD in xenograft sections. There are many CD31 stained micro vessels in DDAH1 xenografts (untreated and mock treated) compared control tissues. In DDAH1 positive xenografts treated with DD1E5, CD31 stained vessels are poorly developed compared to vehicle treated xenografts. From both in vitro and in vivo experiments VEGF expression is correlated with tube formation and MVD, respectively. This shows that DDAH1 inhibition with DD1E5 causes poorer vascularisation in PC3 xenograft tumors (Figure 9B). Till date, large number of anti-angiogenic agents that are tested in clinic are mainly based on either interfering angiogenic ligands or blocking the signals of angiogenic receptors

37-38

. Several anti-

angiogenic agents are still undergoing clinical trials and many cancer types have acquired resistance to anti-angiogenic agents

39

. Successful future developments of anti-angiogenic

therapy require greater understanding of angiogenic signaling pathway and their complex orchestration in tumor vasculature.


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Figure 09: A. To investigate expression of pro angiogenic proteins regulated by DDAH1 and NO in DD1E5 treated xenograft sections, immunohistochemistry was performed. Expression of VEGF, HIF-1α, and NOS is indicated by arrows. DDAH1 expression was also investigated to showing inhibition of DDAH1 curtailed tumor growth not effecting DDAH1 expression where as in DDAH1 knock down tumors its expression is significantly abolished. B. To show effect of DD1E5 on endothelial cells forming micro vessels in xenograft tissues, tumour sections were stained for the endothelial marker CD31 (red colour). DD1E5 repress the micro vessel density in tumors with DDAH1 overexpression compared to untreated and vehicle treated mice. Only representative images of xenograft tumor tissues were shown here.

To understand tumor vasculature, we have taken the advantage of PCa cells expressing distinct phenotypic difference in angiogenesis through stable overexpression of DDAH1 gene. In our previous study, we demonstrated the role of DDAH1 in PCa angiogenesis through the activation of “angiogenic switch” 19. Our results not only provide a molecular basis for the function of DDAH1 in PCa progression, but also suggest promising alternative anti-angiogenic molecular-targeted therapeutics in the treatment of androgen dependent/independent PCa through the inactivation of DDAH1 protein. In conclusion, DD1E5 is found to be potent irreversible inhibitor of human DDAH1. DD1E5 inhibited cellular DDAH1 and leads to inhibit the downstream NO production. Two other compounds (DD1C7 and DD1G7) also have shown better inhibitory potential in cell based assay. These three novel molecules provide basis for developing novel inhibitors against DDAH1 for the control of DDAH1/ADMA pathway to control NO biosynthesis thereby used as antiangiogenic compounds to control tumor angiogenesis.

Material and methods: Materials: All chemicals used in this study were purchased from sigma-aldrich unless otherwise specified.



Chemical libraries for screening: National Mol Bank (NMB) facility has been established at CSIR-Indian Institute of Chemical Technology (IICT) for storing New Molecular Entities (NMEs). Till date, NMB facility stored several novel synthetic/isolated small molecules from synthetic chemists across India and commercial sources. The facility provided compound libraries (10 mM) in 96 well plate format for bioassays.

Typical fluorescent DDAH1 activity assay for primary screening of compounds and orthogonal assay for hDDAH1 inhibition: A fluorescent semi HTS assay was used for screening small molecule libraries against DDAH1 enzyme activity as reported previously with minor modifications. Briefly, enzyme solution was prepared by adding recombinant DDAH1 (0.1μM) enzyme to assay buffer (344 mM KH2PO4, 344 mM KCl, 0.02% Tween- 20 and 4 mM EDTA (pH 8.0)). For screening assay, 45 μl of enzyme solution or assay buffer as control was dispensed into 96 well black polypropylene plate. The stock compounds in DMSO (10mM) were diluted by 10 fold in DMSO and each 0.9 μl of the compound or DMSO as control was dispensed into designated wells containing enzyme solution. The assay plates were briefly centrifuged at 1000g and incubated at room temperature for 10 min. To initiate reaction, 45 μl of Substrate + CPM Solution (7.1 μM CPM, 0.75 μM SMTC, 5 mM KH2PO4, 5 mM KCl, 0.02% Tween-20, pH 2.5) was added to each well and then kept on shaker for 10 sec before recording initial readings. Then the plate was further incubated in incubator with shaking (200 rpm) at 37°c for 45 min. Final readings were taken using a multimode reader (Tecan pro 200) at a excitation and emission wavelength 385ƞm and 480ƞm respectively to measure the product formation. For kinetic assays, fluorescence of the each well was read for every 10 min and reaction rates were estimated from slope by plotting fluorescence versus time. To avoid unnecessary characterization of false positive hit compounds, all primary hits from fluorescence assays were retested for hDDAH1 inhibition by using a previously reported colorimetric orthogonal secondary assay that uses the natural substrate ADMA resulting Lcitrulline product. All reactions were done in triplicates.

Steady-state kinetic studies: A discontinuous colorimetric derivatization of the urea group was used to detect the resulting product L- citrulline, as described previously

40

, and the steady-state catalytic rate


Page 22 of 30

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constants of DDAH-1 catalysed reactions were determined for hydrolysis of

Nω,Nω-

dimethyl-L-arginine (ADMA) and S-methyl-L-thiocitrulline (SMTC). Briefly, various concentrations (10μM to 500μM) of each substrate were treated with enzyme (1 μM), all in KH2PO4 buffer (100 mM) at pH 7.5, 37 °C, and incubated for 30 min after the reaction was stopped with addition of colder reagent and incubate for 15 minutes at 950C and cool it to room temperature for absorbance reading. Control reactions indicate that citrulline production is linear over this time scale. A control reaction without enzyme was also performed at each substrate concentration, enabling background citrulline levels to be subtracted. A citrulline standard curve (0–200 μM), was prepared in the same buffer allowed for quantification of any product absorption observed at 540 ƞm. To obtain steady-state constants, Sigmaplot 12 was used to directly fit observed rates at various substrate concentrations to MichaelisMenten equation.

Docking of chemical structures with hDDAH1: The 3-dimensional X-ray crystallographic structure of DDAH-1 was obtained from the protein data bank (PDB: 3I4A) 6. The protein was prepared and energy was minimized using protein preparation wizard. All the ligands were prepared by Ligprep

41

, the receptor

grid (20x20x20 Å) was generated around N5-(1-iminopropyl)-L-ornithine and the extra precision docking and covalent docking protocol was employed using Glide module of Schrodinger’s LLC. The RMSD of re docked co-crystallized ligand was found to be within the specified range; hence the extra precision docking protocol of GLIDE was employed for current docking studies

42

. The current molecular docking studies were analyzed using

Maestro 11.6.

Mode of inhibition by selected compounds: To examine whether the selected compounds are inhibiting DDAH1 by binding at active site, varying concentrations of selected compounds (0–10μM) and ADMA (25μM to100μM) were mixed in KH2PO4 buffer (100mM) at pH 7.5 and DDAH1 (1μM) is added to start the reaction at 37°C for 30 min and stop the reaction by adding colder reagent then incubate for 15 min at 95°C and cool it to room temperature for absorbance reading (540ƞm). For easy visual interpretation, a Lineweaver-Burk (LB) plot was constructed. To obtain a numerical value for Ki and apparent KM, the initial rate data were fit directly using different inhibition models from SigmaPlot12.



Time and concentration dependent assay: To monitor any time and concentration dependent inactivation of DDAH1 activity with selected compounds DDAH1 (12µM) was incubated with varying concentrations of freshly prepared compounds (60-480µM) in KH2PO4 buffer (100mM) at pH 7.5. At various time points between 0 and 90 min, aliquots of the preincubated mixtures were diluted to 10 fold into a reaction mixture containing ADMA (100µM) and KH2PO4 buffer. The resulting reaction mixtures were incubated at 37°C for 30 min before the product concentrations were determined. At each concentration of selected compounds, remaining activity of DDAH1 was fitted in equation 2. Remaining Activity (%) = 100 × exp(-kobs × t) +C Eq. 2 The pseudo first order inactivation rates and second order rate constant for inactivation were obtained from the equations explained by Lin Hong and Walter Fast 43.

Cell culture conditions: Human prostate cancer PC3, LNCaP cells and other cell lines were purchased from American Type Culture Collection (ATCC) and grown in their respective medium supplemented with 10% fetal bovine serum (FBS), 0.1% streptomycin-penicillin, 1% nonessential amino acids and 1% sodium pyruvate. Cells were incubated at 37°C in a 5% CO2 humidified atmosphere and maintained at sub-confluency by passaging with trypsin-EDTA.

Cell based citrulline assay: Inhibitory activity of hit compounds against intracellular DDAH1 was determined by measuring citrulline produced from ADMA enzyme substrate. The DDAH1 positive PC3 and LNCaP cells were exposed to test compounds at 10μM or vehicle control for 24 hrs. The amount of citrulline generated from ADMA in DDAH1 enzyme reaction was determined according to Knipp et al 40.

Cytotoxicity and cell proliferation assay: Cellular viability and proliferation of DDAH1 positive cells and selected cancer cells exposed to selected compounds was estimated by performing a Sulforhodamine B assay (SRB). The detailed protocol is same as reported recently in KRK Reddy et al 19.

RT-PCR and Western blotting:


Page 24 of 30

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Quantification of mRNA and protein levels of target genes and proteins respectively was performed according to protocols reported in Ummanni et. al

16

. For details such as

primer sequences and antibodies, Supporting table 01 and 02 can be referred.

Estimation of ADMA, IL-8 and bFGF: IL-8, bFGF and ADMA in culture medium collected from DDAH1 positive LNCaP and PC3 cells treated with either hit compounds or vehicle control were estimated by using standard kits (ADMA: Cloud- Clone Corp, IL-8: BioLegend and basic FGF: R&D systems) according to manufacturer’s protocol. To estimate intracellular ADMA, cells were lysed in lysis buffer same as for citrulline assay. The relative levels of measured factors were calculated by normalising to total protein in samples estimated by Bradford reagent.

DDAH1 gene silencing (knockout) using CRSPR-Cas9 system: To Knockout DDAH1 gene in PC3 and LNCaP cells, we use CRISPR-Cas9 gene engineering with cas9 nuclease expression plasmid with puromycin resistance, transactivating CRISPR RNA (tracrRNA) and custom CRISPR RNA (crRNA) designed against target gene site of interest that are purchased from Dharmacon, GE life sciences. All the DNA and RNA components were transfected into cells using DharmaFECT Duo transfection reagent. The target cells (PC3 and LNCaP) were transfected with or without crRNA. After 5h after post transfection media is replaced with fresh medium and cells were allowed to grow for another 24 hr prior to selection with in puromycin (2µg/ml). The selected colonies were allowed to grow in the media without puromycin for one week. Isolated single colonies were propagated further and screened for DDAH1 down regulation by using western blotting.

Tumor regression studies: To analyse the effects of selected compound (DD1E5, 50mg/kg) on DDAH1 inhibition on tumor cells growth in vivo, subcutaneous tumor xenograft model has been used. All animal experiments were performed according to guidelines and requirements of institutional animal ethical committee (IAEC; protocol number: IICT/03/2017). The experiments were performed in 5 groups each consisting 5 male nude mice aged between 4 to 6 weeks (Vivo biotech, India). The xenograft tumor bearing mice were generated as reported by our group recently in KRK Reddy et. al

19

. The five groups were designated as pMSCV

Empty, pMSCV-DDAH1, pMSCV-DDAH1 mock treatment, pMSCV-DDAH1-DD1E5 and CRISPR-CAS9 DDAH1. Once the tumors were grown to 100mm3 size, mice were treated



Page 26 of 30

with either vehicle or test compound DD1E5 (50mg/kg) through intra peritoneum (IP) route twice a week. The tumor measurements were recorded twice weekly using digital Vernier callipers up to 4 weeks, and volume was calculated by equation a (b2)/2

44

, where a and b

represent the length and width of the tumor respectively. After 4 weeks of time, mice were sacrificed by excessive dose of CO2 inhalation and observed for any gross pathological changes in the internal organs. The excised tumors were fixed in formalin and embedded in paraffin blocks. For histopathology and immunohistochemistry, xenograft tissues were cut at 4 μm thickness, stained with haematoxylin and eosin following standard procedure and examined under light microscope (IX71, Olympus, Japan). To determine the proliferative index of the xenograft tumors, mitotic figures from representative tumor sections were measured by using the criteria given by Van Deist et al

45

. Quantitative measurement of

mitotic index was determined using the formula given below. Average number of mitotic figures appeared divided by average total number of cells in 5 microscopic fields multiplied by 100. To determine tissue level expression of DDAH1, VEGF, NOS and HIF-1α in immunostaining was performed using DAB staining as reported previously 19. To observe the vascularisation in xenograft tumors, endothelial specific marker CD31 was stained using its specific antibody and visualized with Alex-555 conjugated secondary antibody. 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑁𝑜.𝑜𝑓 𝑀𝑖𝑡𝑜𝑡𝑖𝑐 𝑓𝑖𝑔𝑢𝑟𝑒𝑠

Mitotic index =

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑡𝑜𝑡𝑎𝑙 𝑛𝑜.𝑜𝑓 𝑐𝑒𝑙𝑙𝑠

× 100 Eq. 3

Notes The authors declare no competing financial interest. Supporting Information: Supporting methods, figures and tables are included in the supporting information Acknowledgments: We thank Ms. Ashita Singh for starting this project and Mr. T. Avinash Raj for technical help in tissue sectioning for histopathology. We thank Science and Engineering Research Board - Department of Science and Technology (DST) for financial support through EMR/2017/001522 and Council for Scientific and Industrial Research (CSIR) under 12th FYP project SMILE (CSC0111). Mr. Karthik Reddy K acknowledges UGC for fellowship. We would also like to express our sincere thanks to Dr. S. Chandrasekhar, Director, CSIR-IICT for support and accesses to National Mol Bank for access to compound library.


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Stone,

E.

M.;

Fast,

W.,

A

continuous

spectrophotometric

assay

for

dimethylarginine

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Novel cellularly active inhibitor regresses DDAH1 induced prostate

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