Further investigation of the potential anti-neoplastic, anti-inflammatory and immunomodulatory actions of phenoxybenzamine using the Broad Institute CLUE platform

Previous clinical studies with the FDA-approved alpha-adrenergic antagonist, phenoxybenzamine, showed apparent efficacy to reverse the symptoms and disabilities of the neuropathic condition, Complex Regional Pain Syndrome; also, the anatomic spread and intensity of this syndrome has a proliferative character and it was proposed that phenoxybenzamine may have an anti-inflammatory, immunomodulatory mode of action. A previous study gave evidence that phenoxybenzamine had anti-proliferative activity in suppression of growth in several human tumor cell cultures. The same report demonstrated that the drug possessed significant histone deacetylase inhibitory activity. Utilizing the Harvard/Massachusetts Institute of Technology Broad Institute genomic database, CLUE, the present study suggests that the gene expression signature of phenoxybenzamine in malignant cell lines is consistent with anti-inflammatory/immunomodulatory activity and suppression of tumor expansion by several possible mechanisms of action. Of particular note, phenoxybenzamine demonstrated signatures that were highly similar to those with glucocorticoid agonist activity. Also, gene expression signatures of phenoxbenzamine were consistent with several agents in each case that were known to suppress tumor proliferation, notably, protein kinase C inhibitors, Heat Shock Protein inhibitors, epidermal growth factor receptor inhibitors, and glycogen synthase kinase inhibitors. Searches in CLUE also confirmed the earlier observations of strong similarities between gene expression signatures of phenoxybenzamine and several histone deacetylase inhibitors.


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A previous study explored a possible anti-proliferative capacity of the drug, 46 phenoxybenzamine (PBZ), as a basis for its apparent therapeutic potential in the treatment of 47 chronic neuropathic pain in the Complex Regional Pain Syndrome (CRPS) (1). The CMap platform showed appreciable similarity to classical histone deacetylase (HDAC) 52 inhibitors. An extensive comparison of PBZ with suberanilohydroxamic acid (SAHA) and 53 trichostatin A (TSA) demonstrated gene expression enrichment scores (connectivity) for PBZ 54 that compared equal to or greater than those for SAHA and TSA for several gene expression 55 signatures in MSigDB that are associated with histone modifications (1). 56 For example, this was true for gene expression signatures from ultraviolet exposure of the 57 epidermis in the 100 to 280 nm wavelength range (UVC class radiation); such exposures are 58 associated with trichothiodystrophy syndrome and xeroderma pigmentosum (4). PBZ also 59 showed highly similar gene expression signatures as those for the histone modifying effects of 60 SAHA and TSA on the viral replication process of human cytomegalovirus; these effects on 61 histones have a "therapeutic" value in the application of oncolytic cancer therapy that is herpes-   It should be noted that the dataset in the earlier online platform of CMap was based almost was only screened with the MCF7 and PC3 cell lines.

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The scoring value obtained in the gene expression analyses is termed "tau;" it ranges from 122 100 to -100 and is a measure of the connectivity between the gene expression signature of the

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A portion of the heatmap output for an inquiry of gene expression signatures with similarities 157 to PBZ is presented in Fig 2. Obviously, PBZ profiled against itself results in the highest score.

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It should be noted that the median tau (connectivity) score for the 9 cell results and the summary 159 is reported in this output presentation. When searching this output in the cloud-based platform,    Each of the perturbagen classes in Table 2 was examined in the Touchstone detailed list for 194 its members; these results are presented in Tables 3, 4, and 5. Only comparisons of PBZ with 195 chemical compounds/drugs (the CP class) were searched in these queries; this represented 2429 196 agents. For these analyses, all agents with connectivity scores above 85 when compared with 197 PBZ were included; for example, in the case of drugs with glucocorticoid agonist activity (Table   198 3) this included 30 of the 44 agents of that perturbagen class with this activity in the Touchstone 199 database.  CSF1R, colony stimulating factor 1 receptor; DDR1, discoidin domain receptor 1; PDGFRA, platelet-derived growth factor receptor-alpha; TIE1, tyrosine kinase with immunoglobulin like and EGF like domains 1; ERBB2, ERBB4, receptor tyrosine kinases; KDR, kinase insert domain receptor; AKT1, serine-threonine protein kinase; NTRK1, neurotrophic tyrosine kinase receptor type 1; MAPK14, mitogen-activated protein kinase 14; NR1I2, nuclear receptor subfamily 1, group I, member 2; PRKCA, PRKCB, PRKCD, PRKCG, protein kinase C alpha, beta, delta, gamma; ALOX5, arachidonate 5-lipoxygenase; GSK3A, GSK3B, glycogen synthase kinase-3 alpha, beta; WEE1, nuclear tyrosine kinase (regulates entry into mitosis); CDK1, CDK2, CDK4, CDK5, CDK9, cyclin-dependent kinase 1, 2, 4, 5, 9; CDK5R1, cyclin dependent kinase 5 regulatory subunit 1; CCNE1, cyclin E1; LCK, lymphocyte specific protein tyrosine kinase; LRRK1, LRRK2, leucine-rich repeat kinase 1, 2; PGR, progesterone receptor; ESR1, estrogen receptor alpha; CYP17A1, CYP2C19, CYP2E1, cytochrome P450 17A1, 2C19, 2E1; NR3C2, mineralocorticoid receptor; CATSPER1, CATSPER2, CATSPER3, CATSPER4, cation channel sperm associated 1, 2, 3, 4; OPRK1, opioid receptor, kappa-1; TRPC5, transient receptor potential channel 5; AR, androgen receptor; SRD5A1, steroid-5-alpha-reductase, alpha. HDAC inhibitors were not represented as a PCL set in the initial output (Table 2), but a 205 search of this class in the detailed list database of Touchstone yielded the connectivities with 206 PBZ for some of these agents ( Table 6). Confirmation of connectivity of PBZ with HDAC 207 inhibitors in the CLUE platform was consistent with our earlier report using the on-line version 208 of CMap, and where PBZ was shown to directly inhibit several HDAC enzymes (1).   agonists; this was completely unanticipated since PBZ shows no resemblance to a steroid 231 chemical structure (Fig 1). There are a total of 44 drugs with glucocorticoid receptor agonist 232 activity in the Touchstone database; the fact that so many individual glucocorticoids had 233 connectivity scores of 85 or greater (Table 3)    Its vasodilating effect is responsible for most of its relatively minor dose-related side effects of 264 hypotension, dizziness, nasal stuffiness and impotence; these effects appear to be self-limiting to 265 some extent with continued intake (14, 15, 23).

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The clinical use of PBZ for its smooth muscle relaxing effects on the urogenital tract is a 267 source of information on the tolerability of the drug with long-term administration. This is found

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Eight gene expression signatures for inhibitors of epidermal growth factor receptor (EGFR) 292 co-sorted strongly with PBZ (Table 4). EGFR inhibition has become a major area of     water retention and weight gain, an effect that is not prominent with bezafibrate (36).

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The second PPAR agonist of recognized clinical importance that matched with PBZ gene

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The three JAK inhibitors in Table 5 that co-sorted with PBZ are experimental agents; they are 367 chemically identified as follows:    (Table 5). HSP90 inhibitors represent a current intense area of clinical investigation as 399 adjunctive agents for the treatment of multiple cancers. Radicicol, the highest scoring agent in relation to PBZ (Table 5) (Table 5).   (Table 5). As with other HIV protease inhibitors, indinavir has a peptide similar 441 structure and is a competitive inhibitor of aspartyl protease. It is currently in clinical use but is 442 used less frequently today since it is not superior to other inhibitors of HIV protease and has 443 more concerns related to renal toxicity (58).
In an attempt to gain some insight into possible reasons for the overlap between gene 445 expression signatures for PBZ and indinavir, indinavir was queried as the index perturbagen in 446 Touchstone. The PCL sets that were returned as important for indinavir relative to the entire 447 database were not similar to any of those for PBZ (Table 2). However, there were two PCL sets 448 for indinavir, "Norepinephrine reuptake inhibitor," and "Tricyclic antidepressant" that had high  The HDAC inhibitor with the highest connectivity score to PBZ was phenylbutyrate (Table The next 4 HDAC inhibitors listed in Table 6

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The present study is an extension of a previous report that suggested that the gene expression