HSP47: Inhibitors

A broad spectrum of molecules has currently been identified with the capacity to modulate the expression or functionality of Hsp47. These molecules not only include natural compounds, peptides and small-molecule chemical inhibitors but also calcium channel blockers, prostaglandin H2 synthase 2 (PGHS-2) inhibitors, and receptor antagonists as well as receptor tyrosine kinase inhibitors (see Table 4).

 

Small-molecule Chemical Inhibitors and Peptides

Hsp47 is an attractive target for the generation of small-molecule inhibitors, as type I procollagen is the only reported target for this chaperone. Four small-molecule chemical inhibitors of Hsp47 have been previously screened from a large chemical library as lead compounds potentially useful for controlling fibrosis and metastasis at low-micromolar concentrations ²³⁸. Pharmacological inhibition of Hsp47 using three of the previously described small-molecule chemical Hsp47 inhibitors was able to reduce the levels of secreted Aβ peptides in AD patients suggesting Hsp47 as being a potential target for preventing the formation and/or growth of amyloid plaques in AD ²²⁴. Two further small-molecule compounds, AK778 and its cleavage product Col003, were screened from chemical libraries using surface plasmon resonance (BIAcore) ²⁴⁴. Both molecules competitively inhibited the interaction of Hsp47 with procollagen and blocked procollagen secretion by destabilizing the procollagen triple helix. Structural information obtained with NMR analysis revealed that Col003 competitively binds to the procollagen-docking site in Hsp47 ²⁴⁴.

Random peptide display libraries were previously used to characterize peptides binding to Hsp47. This approach led to the identification of the LDS affinity peptide (LDSRYSLQAAMYMRM) which mimicks the binding motif of Hsp47 ²⁴⁵.  A novel adenoviral gene delivery system (Ad5-Flag-LDS) was developed and used to transfer the LDS affinity peptide into human head and neck squamous cell carcinoma (HNSCC) cell lines ²⁴⁶. The data raised by Li and co-workers clearly revealed that this gene transfer was suitable to specifically target Hsp47 in HNSCC cells. From these results, one can hypothesize that targeting Hsp47 with Ad5-Flag-LDS might represent a feasible strategy for clinically applicable targeted gene therapy against HNSCC or any Hsp47-positive cancer type ²⁴⁶. More recently, LDS-conjugated superparamagnetic iron oxide (SPIO) nanoparticles were used to successfully target Hsp47 in NIH/3T3 cells stably expressing the Her2/neu receptor (T6-17) ²⁴⁷. After injection of the conjugates into tumor-bearing mice, the comparison of quantitative tumor delivery of SPIO nanoparticles pre-conjugation and post-conjugation to targeting ligand, however, did not elicit a significant difference ²⁴⁷.

 

Natural Compounds and Derivatives

Using in-silico methodology, Pillai et al. revealed the interacting sites of Hsp47 with procollagen and also identified several natural compounds with a potential to bind to this complex such as silymarin, gingerol, resveratrol and curcumin possessing the best affinity towards the binding amino acids of the Hsp47-procollagen complex ²⁴⁸. Silymarin, the polyphenolic fraction from the seeds of Silybum marianum (milk thistle), is a medicinal plant which has been used from ancient times for the treatment of liver and gallbladder diseases. Pillai and co-workers suggested silymarin as a novel therapeutic agent for the management of oral submucous fibrosis (OSF) due to its proposed binding capacity to Hsp47 ²⁴⁸ and the significant association between the up-regulated expression of Hsp47 and type I collagen in lesions of OSF patients ²⁴⁹. In a liver fibrosis model, silymarin as well as lactoferrin were found to significantly decrease Hsp47, TGF-β and elevated liver enzymes; to improve the grade of cirrhosis, and to reduce the deposition of collagen fibers ²⁵⁰. Based on these observations, lactoferrin was considered as being a promising anti-fibrotic drug and inhibitor of Hsp47 ²⁵⁰. Lactoferrin, an iron-binding multifunctional cationic glycoprotein secreted by exocrine glands and by neutrophils, was previously suggested to have a protective effect against liver fibrosis by preventing hepatic stellate cells (HSCs) activation ²⁵¹.

Curcumin, a non-flavonoid polyphenol from turmeric Curcuma longa with anti-tumor capacity, decreased SERPINH1 expression in thioacetamide-induced liver cirrhosis in rats ²⁵². Resveratrol, a dietary compound with anti-cancer properties, down-regulated Hsp47 expression and induced apoptosis in keloid fibroblasts without affecting normal skin fibroblasts ²⁵³. From these findings it can be concluded that resveratrol has therapeutic applicability for treating keloids. Emodin (6-methyl-1,3,8-trihydroxyanthraquinone), isolated from Chinese rhubarb and Japanese knotweed, was previously reported to attenuate bleomycin (BLM)-induced pulmonary fibrosis in mice ²⁵⁴. Now it became evident that emodin reduces levels of Hsp47, TNF-α, IL-6, and TGF-β1 in the lungs of BLM-treated rats via inactivation of Smad-2/3 and STAT-3 ²⁵⁵. Emodin was also noted to mediate a decrease in collagen deposition and suppression of the infiltration of myofibroblasts and inflammatory cells, thereby improving pulmonary function and weight loss as well as preventing death in BLM-treated rats ²⁵⁵.

Gingerol (6-gingerol) is the predominant gingerol in ginger (Zingiber officinale) rhizomes. It has recently been identified by in-silico methodology as a putative interaction partner of Hsp47 ²⁴⁸. Evidence is emerging that gingerol protects against experimental liver fibrosis through blockage of pro-fibrinogenic and pro-inflammatory mediators ²⁵⁶. Xantholipin from Streptomyces flavogriseus is a curved hexacyclic aromatic polyketide anti-tumor antibiotic which was recently identified as a lead drug for the treatment of fibrotic diseases ^{257, 258}. Xantholipin inhibits gene expression of SERPINH1 which is related to a variety of fibrotic diseases ²⁵⁸. Artesunate (butanedioic acid) constitutes a water soluble derivative of artemisinin isolated from the plant Artemisia annua (sweet wormwood), an herb employed in Chinese traditional medicine. Artesunate is a medication used to treat severe forms of Plasmodium falciparum malaria and is considered as being the first-line therapy ²⁵⁹. Recently, artesunate treatment of rats with BLM-induced pulmonary fibrosis yielded anti-fibrogenic effects, including down-regulation of Hsp47, collagen type I, TGF-β1, and Smad-3 ²⁶⁰. In human HSCs, the anti-fibrogenic effects of artesunate could be attributed to the blockage of the FAK/Akt/β-catenin signaling pathway by reducing the levels of phosphorylated FAK, Akt and GSK-3β ²⁶¹.

 

Calcium Channel Blockers and PGHS-2 Inhibitors

Amlodipine (Norvasc®) is a calcium channel blocker used to treat high blood pressure and coronary artery disease. In a mouse model of renal interstitial fibrosis, amlodipine was observed to reduce the fibrotic area in the obstructed kidney and to down-regulate the expression of the SERPINH1 and COL4A1 mRNA possibly by blocking activation of c-Jun N-terminal kinase (JNK) ²⁶². Meloxicam functions as a prostaglandin H₂ synthase 2 (PGHS-2) inhibitor and belongs to the class of non-steroidal anti-inflammatory drugs (NSAIDs). Meloxicam down-regulates the expression of Hsp47 protein and COL4A1 mRNA in mouse renal interstitial fibrosis ²⁶³. Meloxicam was developed for the secondary prevention of cardiovascular events in patients with a history of ischemic stroke or transient ischemic attack ^{264, 265}. Recently, meloxicam has been reported to prevent hypertensive vascular hypertrophy and fibrosis in rats accompanied by a suppresion of Hsp47 protein and TGFB1 mRNA expression ²⁶⁶.

 

Receptor Antagonists and Receptor Tyrosin Kinase Inhibitors

Nintedanib (Ofev®) is a receptor tyrosine kinase inhibitor of platelet-derived growth factor receptor (PDGFR), fibroblasts growth factor receptor (FGFR), and vascular endothelial growth factor receptor (VEGFR) which was first developed as an anti-cancer drug. All of  the mediators mentioned are crucially involved in the pathogenesis of IPF ²⁶⁷. Nintedanib was approved for IPF therapy as the drug has been determined to decelerate disease progression. Nintedanib represents a safe and commonly well tolerated treatment for IPF when used in routine practice ^{268, 269}. Most recently, nintedanib was identified to down-regulate TGF-β1-induced transcription of COL1A1, COL3A1, and FN1 and also to decrease basal levels of SERPINH1 mRNA in IPF fibroblasts ²⁷⁰. The inhibitor consistently down-regulated transcript and protein levels of basal and TGF-β1-induced procollagen I and attenuated the secretion of procollagen I and III in these cells. Remarkably, nintedanib suppressed collagen type I fibril formation and reduced formation and changed appearancee of collagen fibril bundles, exhibiting a novel mechanism of action for this anti-fibrotic agent ²⁷⁰.

Terutroban (1-naphthalene-propanoic acid) represents a specific thromboxane/prostaglandin endoperoxide receptor (TPr) antagonist which shows anti-thrombotic, anti-vasoconstricting, and anti-atherosclerotic characteristics ²⁷¹. Terutroban was found to prevent hypertensive vascular hypertrophy and fibrosis in rats by blocking the expression of Hsp47 protein and TGFB1 transcripts ²⁶⁶. Recently, terutroban diminished liver fibrosis and down-regulated mRNA expression of COL1A1 and TGFB1 as well as protein expression of α- smooth muscle actin (α-SMA), without affecting the eNOS pathway ²⁷².

 

Others

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone, Esbriet®) is a new anti-fibrotic, anti-inflammatory and anti-oxidant drug that blocks the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). In vitro studies of normal human lung fibroblasts demonstrated that pirfenidone abolishes TGF-β1-induced collagen synthesis by blocking the up-regulation of SERPINH1 and COL1A1 mRNA ²⁷³. Quantitative analysis of primary human lung fibroblasts (phLF) isolated from human IPF patients revealed a down-regulation of TGF-β1-induced COL5A1 and FN1 mRNA as well as a decline in basal levels of SERPINH1 transcripts in IPF fibroblasts ²⁷⁰. Moreover, pirfenidone slightly decreased TGF-β1-induced COL1A1 expression without affecting procollagen I protein synthesis and secretion. In contrast to nintedanib, pirfenidone only slightly down-regulated COL3A1 transcripts while the production and secretion of procollagen type III remained almost unaffected. The drug nonetheless suppressed extracellular fibril formation and reduced assessment of spontaneous fibril assembly of type I collagen, similarly to nintedanib ²⁷⁰. Interestingly, both pirfenidone and nintedanib considerably down-regulated type V collagen in IPF fibroblasts as demonstrated by Knüppel and collaborators. Collagen V, a minor component of collagen type I fibrils, was discovered to crucially impact the initiation of the collagen fibril assembly and the regulation of the fiber size ²⁷⁴. Pirfenidone has been approved for the treatment of IPF in many countries, including the European Union (EU), the USA and Japan. The safety and tolerability of pirfenidone have been well characterized in clinical trials ^275-277. New evidence has emerged to demonstrate that pirfenidone provides a survival benefit in a real-life IPF cohort together with a lower rate of drug discontinuation due to photosensitivity reactions ²⁷⁸.

Although targeting Hsp47 in certain pathologies represents an attractive approach in disease management, one should consider that systemic administration of inhibitors to collagen chaperones posses systemic risks, particularly in tissues with high normal expression of type I collagen, like skin and bone. Therefore, alternative approaches such as introduction of miR-29 and silencing of Hsp47 have been developed and conducted. One such approach utilized siRNA against SERPINH1 aiming to abolish fibrosis in animal models ²¹⁶. Sato et al. conjugated vitamin A to liposomes carrying siRNAs targeting gp46, a rat ortholog of Hsp47 ²¹⁶. Evaluation of liposome conjugates showed uptake predominantly in livers, demonstrating organ specificity. siRNA-bearing vitamin A-conjugated liposomes rapidly resolved fibrosis and prolonged survival in rats with dimethylnitrosamine-induced cirrhosis ²¹⁶. These data represent an exciting advance in siRNA-mediated treatment of fibrosis and demonstrate that targeting collagen production in this way might be considered as being an effective anti-fibrotic strategy. With respect to cancer, transfection of cervical squamous cell carcinoma cells using siRNA against SERPINH1 almost completely repressed expression of Hsp47 at both, the mRNA and protein level ¹⁸³. In line with this notion, transfection of HeLa cells with miR-29a significantly repressed SERPINH1 mRNA and Hsp47 protein expression in these cells. However, further studies aiming to assess the functional consequences of Hsp47 disruption and/or modulation in certain clinical settings are warranted and will help to shed light on the molecular mechanisms and the versatility of Hsp47 targeting approaches.

 

Table 4: Inhibitors and Modulators of Hsp47 (Serpin H1)

Agent	References
Natural compounds and derivatives
Artesunate (butanedioic acid)	Wang et al. (2015) 260, Lv et al. (2018) 261
Curcumin	Pillai et al. (2014) 248, Ali et al. (2014) 252
Emodin (6-Methyl-1,3,8-trihydroxyanthraquinone)	Chen et al. (2009) 254, Guan et al. (2016) 255
Gingerol	Pillai et al. (2014) 248, Algandaby et al. (2016) 256
Lactoferrin	Tung et al. (2014) 251, Rizk et al. (2018) 250
Resveratrol	Ikeda et al. (2013) 253, Pillai et al. (2014) 248
Silymarin	Pillai et al. (2014) 248, Rizk et al. (2018) 250
Xantholipin	Terui et al. (2003) 258, Winter et al. (2013) 257
Small-molecule chemical inhibitors
4 Compounds	Thomson et al. (2005) 238
AK778, Col003	Ito et al. (2017) 244
Peptides
LDSRYSLQAAMYMRM	Li et al. (2008) 246, Elias et al. (2014) 247
Calcium channel blockers
Amlodipine (Norvasc®)	Honma et al. (2016) 262
Non-steroidal anti-inflammatory drugs (NSAIDs)
Meloxicam	Honma et al. (2014) 263
Thromboxane/prostaglandin endoperoxide receptor antagonists
Terutroban (1-naphthalene-propanoic acid)	Chamorro et al. (2009) 271, Gelosa et al. (2014) 266
Receptor tyrosine kinase inhibitors
Nintedanib (Ofev®)	Wollin et al. (2015) 267, Hughes et al. (2016) 269, Knüppel et al. (2017) 270, Fletcher et al. (2018) 268
Miscellaneous uncategorized agents
Pirfenidone (Esbriet®) 5-methyl-1-phenyl-2-(1H)-pyridone	Nakayama et al. (2008) 273, King et al. (2014) 277, Lancaster et al. (2016) 276, Cottin et al. (2018) 275