Chapter 4: Advanced Connectivity Map analysis for identifying DCUN1D1 inhibitors using
4.5 Results
We performed CMap analysis using the proteins significantly differentially expressed following quantitative SILAC proteomics analysis of DCUN1D1 knockdown in DU145 PCa cell lines. We found 33 proteins to be significantly differentially expressed in sample LH2 with 14 downregulated and 19 upregulated as well as 17 proteins which were significantly differentially expressed in sample LH3, with 8 downregulated and 9 upregulated. We queried the samples in the CMap database using the
“query app”, according to gene name, with a final number of 14 downregulated and 17 upregulated proteins in sample LH2, which were identified by the database as valid genes for analysis as part of the database BING space. In terms of sample LH3, we had 9 upregulated valid gene name identifications and 8 downregulated valid gene name identifications, which were below the threshold of 10 required for CMap analysis. We therefore included the protein/gene name identification that was nearest to the significant differential expression cut-off (1 standard deviation below the mean), which was P4HB in the upregulated list as well as CDK1, because one of the gene name identifications in the upregulated list was not found within the BING space. Additionally, we included NAA15 and PHGDH in the downregulated list and computed the CMap analysis.
We obtained the query results in the form of a heatmap and detailed tabulated list. We initially performed the analysis by viewing the CMap signatures that persist across the cell lines represented in the database (Figure 41). It is a perturbagen-centric summarization of the data across the 9 cell lines examined in the database, based on the connectivity score of each perturbagen per cell line and summarized across the cell lines. The connectivity score is a measure between -100 and 100, with CMap signatures close to 100 showing strong positive connectivity and those close to -100 showing strong negative connectivity or opposition. We obtained outputs based on the whole reference dataset (Touchstone) which included all the perturbagens (knockdown, overexpression and compounds) and focused our analysis on the outputs obtained based on the compound perturbagen type. Significantly, of the top 22 compounds observed, we found the following compounds in common between the samples: BI-2536 (PLK inhibitor/apoptosis stimulant/cell cycle inhibitor), NU-7441 (DNA dependent protein kinase inhibitor) and XMD-1150 (leucine rich repeat kinase inhibitor). Interestingly, in sample LH2, we found the androgen receptor agonist testosterone and docetaxel, the tubulin inhibitor and current therapeutic agent for PCa treatment. Additionally, we found calpeptin in sample LH3, which is has been described to be a proteasome inhibitor.
109
Figure 41. Showing the top ranked CMap compound signatures based on connectivity score.
Heatmaps showing the 9 core cell lines of the reference dataset and the rank-ordered list of compounds for sample LH2 (top) and sample LH3 (bottom).
110
We then performed further analysis of the dataset by selecting for the outputs based on the PCa cell lines within the 9 core cell lines in the CMap database whose cellular signatures were connected to that of the DCUN1D1 differential expression signature (Table 18 and 19). We obtained outputs for both samples and observed that the compounds in common between sample LH2 and LH3 with high connectivity scores (>90) in the PC3 cell line CMap signatures were: talniflumate (cyclooxygenase inhibitor), atorvastatin (HMGCR inhibitor), GR-135531 (melatonin receptor agonist), W-9 (calmodulin antagonist), RX-821002 (adrenergic receptor antagonist) and palmitoylethanolamide (cannabinoid receptor agonist). Additionally, we found ISOX (HDAC inhibitor), ergocryptine (dopamine agonist), XMD-885 (leucine rich repeat kinase inhibitor), mestanolone (androgenic steroid), XMD-892 (MAP kinase inhibitor), carmoxirole (dopamine receptor agonist), and VX-222 (HCV inhibitor) in common in the VCap cell line CMap signatures. Significantly, we found podophyllotoxin and thapsigargin to score highly in the PC3 cell line signature, in sample LH2 and LH3, respectively. These compounds were found previously following CMap analysis of knockdown of DCUN1D1 in PCa and DNA microarray analysis (Vava and Zerbini, 2014). Thapsigargin was found to decrease mRNA expression of DCUN1D1, while, podophyllotoxin was found to decrease mRNA and protein expression of DCUN1D1 and to have an additive inhibitory effect on PCa proliferation in combination with monensin (Vava and Zerbini, 2014).
Lastly, we analysed the data based on perturbagen class (Figure 42). This was defined as a “group of compounds that have the same annotated mechanism of action or genetic perturbagens that are part of the same gene family or are targeted by the same compound”. Of the signatures with the highest connectivity scores we found the following perturbagen classes: leucine rich repeat kinase inhibitor, PKC activator, DNA dependent protein kinase inhibitor, bromodomain inhibitor, IGF-1 inhibitor, bile acid and HMGCR inhibitor in common across the samples.
111
Table 18. Showing the top ranked CMap compound signatures based on connectivity score (> 90) in PC3 in sample LH2 and LH3.
LH2 - PC3 LH3 - PC3
Rank Score Name Description Rank Score Name Description
20 99.7 HSP90-inhibitor HSP inhibitor 16 99.51 RX-821002 Adrenergic receptor antagonist
26 99.42 Talniflumate Cyclooxygenase inhibitor 26 99.24 Talniflumate Cyclooxygenase inhibitor
51 98.06 Danusertib Aurora kinase inhibitor 32 99.09 W-9 Calmodulin antagonist
61 97.25 BIIB021 HSP inhibitor 33 98.94 Phenanthridone PARP inhibitor
71 96.82 K3644 Kinesin-like spindle protein inhibitor 38 98.83 Cycloserine Bacterial cell wall synthesis inhibitor
79 96.41 Metoclopramide Dopamine receptor antagonist 48 98.31 XAV-939 Tankyrase inhibitor
84 96.23 ST-91 Adrenergic receptor agonist 50 98.26 Olaparib PARP inhibitor
93 95.88 Atorvastatin HMGCR inhibitor 60 97.99 GR-135531 Melatonin receptor agonist
99 95.5 Fluorometholone Glucocorticoid receptor agonist 77 97.18 Sulmazole Adenosine receptor antagonist
107 95.24 Zardaverine Phosphodiesterase inhibitor 78 97.1 Taurocholic-acid Bile acid
115 94.69 PCO-400 Potassium channel activator 79 97.08 Spectinomycin Bacterial 30S ribosomal subunit inhibitor
119 94.43 GR-135531 Melatonin receptor agonist 80 97.04 Fostamatinib SYK inhibitor
129 93.51 Heraclenol Vitamin K antagonist 82 96.84 NBI-27914 CRF receptor antagonist
130 93.46 PD-123319 Angiotensin receptor antagonist 86 96.49 QL-X-138 MTOR inhibitor
136 93.13 W-9 Calmodulin antagonist 89 96.23 PI-828 PI3K inhibitor
141 92.66 RX-821002 Adrenergic receptor antagonist 98 95.82 LY-288513 CCK receptor antagonist
145 92.22 Podophyllotoxin Microtubule inhibitor 121 94.18 WYE-354 MTOR inhibitor
147 92.07 PJ-34 PARP inhibitor 122 94.12 PP-1 SRC inhibitor
156 91.51 Hexylresorcinol Local anesthetic 123 94.1 Methyllycaconitine Acetylcholine receptor antagonist
164 90.69 Palmitoylethanolamide Cannabinoid receptor agonist 131 93.77 PCO-400 Potassium channel activator
168 90.33 Antimycin-a ATP synthase inhibitor 141 93.05 Oxybenzone Lipase inhibitor
144 92.92 AMN-082 Glutamate receptor modulator 148 92.55 Thapsigargin ATPase inhibitor
150 92.42 Colforsin Adenylyl cyclase activator 151 92.41 Atorvastatin HMGCR inhibitor 153 92.3 Givinostat HDAC inhibitor
160 91.57 Hispidin PKC inhibitor
161 91.56 SC-19220 Prostanoid receptor antagonist 164 91.38 Forskolin Adenylyl cyclase activator
169 90.92 Skatole Thrombin inhibitor
170 90.8 GR-113808 Serotonin receptor antagonist 175 90.43 Terconazole Sterol demethylase inhibitor 176 90.4 Oxprenolol Adrenergic receptor antagonist 177 90.37 Palmitoylethanolamide Cannabinoid receptor agonist 180 90.3
Phosphodiesterase-V-
inhibitor-II Phosphodiesterase inhibitor
112
Table 19. Showing the top ranked CMap compound signatures based on connectivity score (> 90) in VCap in sample LH2 and LH3.
LH2 - VCap LH3 - VCap
Rank Score Name Description Rank Score Name Description
22 98.8 ISOX HDAC inhibitor 3 99.82 VX-222 HCV inhibitor
25 98.52 Ergocryptine Dopamine agonist 12 99.39 ISOX HDAC inhibitor
34 97.78 XMD-885 Leucine rich repeat kinase inhibitor 14 99.28 BI-2536 PLK inhibitor
35 97.76 Mestanolone Androgenic steroid 17 99.1 Carmoxirole Dopamine receptor agonist
36 97.75 Methandriol Androgenic steroid 19 98.95 XMD-892 MAP kinase inhibitor
43 97.48 Carbamazepine Carboxamide antiepileptic 32 97.83 XMD-885 Leucine rich repeat kinase inhibitor
49 97.25 Norethisterone Progesterone receptor agonist 35 97.46 PI-828 PI3K inhibitor
50 97.2 Exemestane Aromatase inhibitor 36 97.4 Ergocryptine Dopamine agonist
54 97.05 Formestane Aromatase inhibitor 44 96.85 LY-303511 Casein kinase inhibitor
62 96.65 XMD-892 MAP kinase inhibitor 51 96.08 Epinephrine carbonic anhydrase activator
82 94.79 Carmoxirole Dopamine receptor agonist 59 95.41 Androstenedione Cytochrome P450 inhibitor
83 94.77 Testosterone Androgen receptor agonist 60 95.35 XMD-1150 Leucine rich repeat kinase inhibitor
84 94.68 VX-222 HCV inhibitor 61 95.3 Chlorambucil DNA inhibitor
85 94.68 ZM-39923 JAK inhibitor 62 95.25 Tretinoin Retinoid receptor agonist
89 94.58 Skatole Thrombin inhibitor 64 95.13 Chromanol Potassium channel blocker
94 94.25 Noretynodrel Progestogen hormone 66 94.98 Irinotecan Topoisomerase inhibitor
96 94.03 Gestrinone Progesterone receptor antagonist 74 93.92 Fostamatinib SYK inhibitor
98 93.79 Vorinostat HDAC inhibitor 78 93.74 JWE-035 Aurora kinase inhibitor
101 93.59 Dovitinib EGFR inhibitor 84 92.98 Trimethobenzamide Histamine receptor antagonist
103 93.39 JLK-6 Gamma secretase inhibitor 90 92.39 QS-11 ARFGAP inhibitor
105 93.14 Flubendazole Tubulin inhibitor 93 92.29 L-690488 Inositol monophosphatase inhibitor
108 92.99 CD-437 Retinoid receptor agonist 108 91.26 Mestanolone Androgenic steroid
109 92.97 Enobosarm Androgen receptor modulator 111 91.2 Pyroxamide HDAC inhibitor
113 92.74 Linifanib PDGFR receptor inhibitor 113 90.79 CAY-10577 Casein kinase inhibitor
114 92.73 Aminolevulinic-acid Oxidizing agent 114 90.75 Midazolam Benzodiazepine receptor agonist
115 92.61 KU-0060648 DNA dependent protein kinase inhibitor 115 90.75 Alprazolam Benzodiazepine receptor agonist
122 92.12 MNITMT Lymphocyte inhibitor 120 90.29 r(-)-propylnorapomorphine Dopamine receptor agonist
124 91.8 Flurofamide Urease inhibitor 121 90.17 DR-2313 PARP inhibitor
130 91.49 Fluorometholone Glucocorticoid receptor agonist 122 90.1 Everolimus MTOR inhibitor
146 90.4 Docetaxel Tubulin inhibitor
149 90.16 Norethindrone Progesterone receptor agonist
113
Figure 42. Showing the top ranked CMap signatures based on connectivity score and perturbagen class. Heatmaps showing the 9 core cell lines of the reference dataset and the rank-ordered list of compounds in sample LH2 (left) and sample LH3 (right).
114