Science

GSNOR Inhibition as a Pleotropic Therapy for Inflammatory, Oxidant-based, and Fibrotic Diseases

As Charles Darwin published 157 years ago in “On the Origin of Species”, evolution through natural selection has produced the amazing phenotypic diversity of living organisms on Earth. Evolution has also shaped the molecular and cellular systems that regulate the physiology of all those species. Some systems are very different—photosynthesis in plants, which is absent in animals. However, other systems, particularly signaling systems, are very similar between species. One prominent example is the nitrosylation signaling system which has been in evolution for a billion years. For example, nitrosylation is important in the multi-competent cells in the hemolymph of an arthropod--the American horseshoe crab (Limulus polyphemus), an animal which has changed little over 500 million years of evolution [1] and still depends upon its nitrosylation pathways. All other eukaryotes alive today depend upon this system as well for a diverse set of physiological responses, many of which cause disease when mis-regulated.

The importance of nitrosylation in physiology and medicine was discovered in 1977 when nitric oxide (NO), a gas, was discovered to have important signaling roles in many different cells and organs. That discovery resulted in the awarding of Nobel Prizes in 1998 to Ferid Murad, Robert F. Furchgott and Louis J. Ignarros. Dr. Murad joined SAJE’s scientific advisory board in December of 2015 and both supports and plays a key role in our programs.

Given the fact that nitrosylation, as a physiological regulator, has been conserved in evolution for so long implies that it must play important roles in maintaining organismal homeostasis or it would have been eliminated by evolution if it produced toxic consequences. Those roles are expected to be significant and diverse because evolution tends to reutilize a signaling pathway for multiple applications. SAJE, based on its data and surveys of the relevant literature, believes that nitrosylation plays major pleiotropic roles in regulating multiple, important therapeutic pathways. To date, those expectations have been realized.

As support for these hypotheses, SAJE Pharma and its collaborators have demonstrated that inhibiting the enzyme S-nitrosoglutathione reductase (GSNOR) increases the levels of nitrosylation on critical proteins and produces numerous therapeutic benefits that are relevant to many important diseases in various organ systems. GSNOR is a reductase which breaks down or reduces S-nitrosoglutathione (GSNO) which is the stable, cellular storage form of NO. NO is extremely reactive with a tissue half-life of seconds and limited tissue penetration, whereas GSNO has a half-life of about 5.5 hrs and extensive cellular and plasma distribution. Thus, by inhibiting the breakdown of GSNO by inhibiting GSNOR, the cellular levels of GSNO increase which trans-nitrosylate critical proteins that activate evolutionarily selected therapeutic pathways. Among those pathways and benefits are:

  1. Anti-inflammation: reduction in the number of eosinophils and lymphocytes that infiltrate inflamed tissue; inhibition of ICAM-1; inhibition of the cytokines: IFN-γ, TNF-α, TGF-β, IL-4, IL-5, IL-6, IL-12(p40), IL-12(p70), IL-13, Il-17, and IL-23 [References 2, 3, 4 & SAJE unpublished].
  2. NFĸB: Inhibition of the activation of NFĸB by increasing the S-nitrosylation of Iĸĸβ, which inhibits its kinase activity and suppresses NFĸB activation [5] and, in turn, decreases the expression of inflammatory genes.
  3. Inhibition of the chemokines CCL-2 (MCP-1) and CCL11: CCL-2 recruits monocytes, memory T cells, and dendritic cells to the sites of inflammation produced by either tissue injury or infection. CCL-11 selectively recruits eosinophils by inducing their chemotaxis, and therefore, is implicated in allergic responses. Both are involved in induction of fibrosis in various tissues [2,3].
  4. Anti-Oxidant: induction of Nrf2/ARE system of anti-oxidant enzymes to inhibit the production of reactive oxygen species (ROS) [6], which are causative in the induction of fibrosis [ 7,8].
  5. Anti-fibrotic: SPL-334 not only prevents progression of fibrosis, but also reverses existing bleomycin induced fibrosis [3]--due to inhibition of ROS, CCL-2, CCL-11, and Connective tissue Growth Factor (CTGF). Reversal of existing fibrosis is almost unprecedented among clinical candidates. SPL-891.1 prevents NASH in a mouse model of the disease.
  6. EMT: Attenuation of epithelial-mesenchymal transition (EMT) as measured by decreased TGF-β induced collagen synthesis in human fibroblast cells, in vitro [3].
  7. Bronchodilation through opening of constricted bronchioles [2,4].
  8. Increased mucus clearance [2].
  9. Activation of Soluble Guanylyl Cyclase (sGC) which regulates the cyclic guanosine monophosphate (cGMP) system [5]. cGMP acts as a second messenger much like cyclic AMP. Its mechanism of action is activation of intracellular protein kinases in response to the binding of membrane impermeable peptide hormones to the external cell surface.

GSNOR inhibition increases the nitrosylation of evolutionarily selected signal transduction proteins by increasing the cellular concentration of S-nitrosoglutathione (GSNO) which trans-nitrosylates accessible regulatory cysteines. Trans-nitrosylation by GSNO proceeds by a different chemical mechanism than nitrosylation by NO. GSNO does not produce NO as some literature suggests. Furthermore, GSNOR inhibition does not cause nitrosative stress, but rather, prevents it [6,9,10]. This lack of nitrosative stress is a clear advantage for using GSNOR inhibition rather than exogenous NO itself or non-specific NO donors to effect the therapeutic benefits of nitrosylation.

By inhibiting GSNOR, we simultaneously activate many signal pathways in a natural and balanced way to promote multi-faceted therapeutic outcomes, as noted above. This balanced approach is in contrast to drugs that knock down a single target that is disease related—TNF-α, TGF-β, IL-6 and many others. We believe our GSNOR inhibition approach is more efficacious and also safer because we are simultaneously regulating many disease processes in a balanced way.

In summary, we believe SAJE Pharma’s data suggest that GSNOR inhibition represents a new paradigm in the pharmacological therapy of many human and animal diseases. The pleiotropic effects that result from inhibiting GSNOR are able to simultaneously regulate multiple disease pathways in a controlled evolutionarily selected way that avoids toxicity. Thus, GSNOR inhibition by small molecules provides powerful synergistic therapies for many important diseases, with so far limited, to no, toxicity.

References
  1. Ottaviani, E., Paemen, L.R., Cadet, P. and Stefano, G.B. Evidence for nitric oxide production and utilization as a bacteriocidal agent by invertebrate immunocytes (1993) Eur. J. Pharmacol 248, 319-324
  2. Ferrini, M. E., Simons, B. J., Bassett, D. J., Bradley, M. O., Roberts, K., and Jaffar, Z. (2013) S-nitrosoglutathione reductase inhibition regulates allergen-induced lung inflammation and airway hyperreactivity, PLoS One. 2013 Jul 25;8(7):e70351. doi: 10.1371/journal.pone.0070351. Print 2013.
  3. Luzina, I., Lockatell, V.,Todd, N., Kopach, P., Pentikis, H., Atamas, S. Pharmacological in vivo inhibition of S-nitrosoglutathione reductase attenuates bleomycin-induced inflammation and fibrosis. JPET Fast Forward. Published on July 24, 2015 as DOI: 10.1124/jpet.115.224675
  4. Loretta G. Que, Matthew W. Foster, Erin N. Potts, Erik J. Soderblom, Zhonghui Yang, David M. Gooden, M. A. Moseley, and W M. Foster. Systemic And/Or Local Aerosol Inhibition Of S-Nitrosoglutathione Reductase (GSNOR) Ameliorates Physiologic, Biologic, And Proteomic Phenotypes In An Allergic Mouse Model Of Inflammatory Airway Disease. Chapter DOI: 10.1164/ajrccm-conference.2011.183.1_MeetingAbstracts.A407510. GSNORi in their abstract and in #5 below is the same compound as SPL-334
  5. Sanghani, P.C., et al., Kinetic and cellular characterization of novel inhibitors of S-nitrosoglutathione reductase. J Biol Chem, 2009. 284(36): p. 24354-62.
  6. Foster, M. W., Yang, Z., Gooden, D. M., Thompson, J. W., Ball, C. H., Turner, M. E., Hou, Y., Pi, J., Moseley, M. A., and Que, L. G. (2012) Proteomic characterization of the cellular response to nitrosative stress mediated by s-nitrosoglutathione reductase inhibition, J Proteome Res 11, 2480-2491.
  7. Artaud-Macari, E, Goven, D, Brayer, S, Hamimi, A, Besnard, V, Marchal-Somme, J, Al,i ZE, Crestan,i B, Kerdine-Romer, S, Boutten, A and Bonay, M (2013) Nuclear factor erythroid 2-related factor 2 nuclear translocation induces myofibroblastic dedifferentiation in idiopathic pulmonary fibrosis. Antioxidants & redox signaling 18:66-79.
  8. Hecker, L., Vittal, R., Jones, T., Jagirdar, R., Luckhardt, T., Horowitz, J., Pennathur, S., Martinez, F., and Thannickal, V. NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury Nature Medicine 15, 1077 - 1081 (2009).
  9. Taylor, B.S., L.H. Alarcon, and T.R. Billiar, Inducible nitric oxide synthase in the liver: regulation and function. Biochemistry (Mosc), 1998. 63(7): p. 766-81.
  10. Assreuy, J., et al., Feedback inhibition of nitric oxide synthase activity by nitric oxide. Br J Pharmacol, 1993. 108(3): p. 833-7.

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