SAJE White Papers
1. GSNOR Inhibition Does NOT Cause Nitrosative Stress
Matt Bradley, PhD
SAJE Pharma, LLC
The enzyme S-nitrosoglutathione reductase (GSNOR) in the presence of NADH reduces S-nitrosoglutathione (GSNO) to glutathione disulfide and hydroxyl amine. Inhibition of GSNOR activity by a SAJE drug prevents the breakdown of GSNO and causes an increase in its cellular pool, which can then increase the level of cysteine nitrosylation on proteins that are in the tightly coupled evolutionarily selected nitrosylation pathways. GSNOR inhibition does NOT increase the amount of free nitric oxide (NO), a radical which, in too high concentrations, can cause toxicity by nitrosative stress. In contrast, it reduces NOS2 and eNOS activity and the production of NO by feedback inhibition by NO and GSNO. The evidence that GSNOR inhibition does not cause nitrosative stress is the following:
- GSNO feeds back and inhibits the inducible nitric oxide synthase (NOS2), which reduces the amount of free NO and thus reduces nitrosative stress (Br. J. Pharmacol. (1993), 108, 833-837. Feedback inhibition of nitric oxide synthase activity by nitric oxide. Assreuy, F.Q. Cunha, F.Y. Liew & S. Moncada
- The expression of several cytokine-inducible immunomodulators, including osteopontin, cyclooxygenase-2, and nitric oxide synthase isoform 2 (NOS2), were decreased by SPL-334 (GSNORi in the paper) in cultured cells. Thus, GSNOR inhibition by SPL-334 does not increase nitrosative stress—rather it reduces it. [Proteomic Characterization of the Cellular Response to Nitrosative Stress Mediated by S-Nitrosoglutathione Reductase Inhibition. Matthew W. Foster,, Zhonghui Yang, David M. Gooden, J. Will Thompson, Carol H. Ball,Meredith E. Turner, Yongyong Hou, Jingbo Pi, M. Arthur Moseley, and Loretta G. Que. J. Proteome Res. dx.doi.org/10.1021/pr201180m | J. Proteome Res. 2012, 11, 2480−2491]
- GSNOR knockout (-/-) mice develop, grow, behave, reproduce, and survive very similarly to normal mice. Thus, complete absence of GSNOR from conception to death does not cause toxicity of the sort that extensive nitrosative stress would cause. [Liu L, Yan Y, Zeng M, Zhang J, Hanes MA, Ahearn G, McMahon TJ, Dickfeld T, Marshall HE, Que LG, Stamler JS (2004). Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell 116:617-628]
- GSNOR (+/-) mice are also apparently normal. They are analogous to what pharmacological inhibition of GSNOR would be for even chronic dosing for a part of the lifespan, since small molecule enzyme inhibitors in general do not totally obliterate an enzyme, but just reduce its activity which is the goal of GSNOR inhibition therapy. The fact that GSNOR (+/-) mice have normal physiology, lifespan, and behavior indicate that they do not suffer from nitrosative stress.
- SPL-334 in cell culture is not toxic at doses up to 100uM and with 500uM GSNO in the medium causing an increase in nitrosylated proteins. If GSNOR inhibition caused toxic nitrosative stress, it would have done so under those conditions, but did not. [“Kinetic and Cellular Characterization of Novel Inhibitors of S-Nitrosoglutathione Reductase.” Paresh C. Sanghani, Wilhelmina I. Davis, Sharry L. Fears, Scheri-Lyn Green, Lanmin Zhai, Yaoping Tang, Emil Martin, Nathan S. Bryan, and Sonal P. Sanghani. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 36, pp. 24354–24362, September 4, 2009]
- Nitrosylation control of cellular pathways has been used in biology since the evolution of eukaryotes. If nitrosylation as a process had significant toxicities associated with it, it would have been eliminated from cellular physiology by evolution. The goal of GSNOR inhibition therapy is not to eliminate GSNOR, but rather to reduce GSNOR activity enough to increase nitrosylation of those protein pathways that produce therapeutic advantages in different diseases. So far we have identified those therapeutically relevant pathways as: anti-inflammation (SPL-334 decreases ICAM-1, NFĸB and the cytokines: Th1 (IFN-γ, TNF-α) and Th2 (IL-4, IL-5, IL-6, IL-12(p40), IL-12(p70), and IL-13, the chemokine CCL 11, and the number of BALF eosinophils and lymphocytes); oxidant damage control by induction of Nrf-2 anti-oxidant enzymes; and fibrosis remodeling. Such pleotropic multiple therapeutic efficacies by inhibiting one enzyme with small molecules are unprecedented in pharmacology.
- S–nitrosylation of HSP90 on a susceptible cysteine in the C-terminal domain of the protein causes HSP90 to interact with and inhibit the enzymatic activity of the endothelial nitric oxide synthase (eNOS). This finding represents an autoregulatory feedback mechanism by which excess NO or GSNO checks possible nitrosative stress. [S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities Antonio Martı´nez-Ruiz, Laura Villanueva, Cecilia Gonza´lez de Ordun, Daniel Lo´ pez-Ferrer, Marı´a A´ ngeles Higueras, Carlos Tarı´n, Ignacio Rodrı´guez-Crespo, Jesu´ s Va´ zquez, and Santiago Lamas. PNAS,June 14, 2005 vol. 102 no. 24 8525– 8530.]
- H2S increases eNOS dimerization, whereas NO decreases eNOS activity by promoting the formation of eNOS monomers. This finding represents yet another mechanism for feedback inhibition of NOS activity by NO and GSNO, since GSNO nitrosylates as does NO. [Zaid Altany, YoungJun Ju, GuandungYanf, and Rui Wang. The coordination of S-sulfhydration, S-nitrosylation, and phosphorylation of endothelial nitric oxide synthase by hydrogen sulfide. Sci. Signal. 2014, vol. 7, issue 342: ra 87.]
2. Safety of GSNOR Inhibition
The following White Paper summarizes the existing knowledge about the safety of using, for therapeutic purposes, inhibitors of the enzyme S-nitrosoglutahione reductase (GSNOR).
Two fundamental types of toxicity:
Mechanistic toxicity: In which the inhibition or activation of the basic mechanism of drug action causes both a therapeutic effect and also a toxic effect. If the therapeutic effect occurs at lower concentrations than the toxic effect, then the drug might be used to treat a disease. However, if the therapeutic and toxic effects occur at concentrations that are too close to each other, then the drug may be too dangerous to use. In particular, some patients may metabolize the drug slower than the majority of patients and thus be exposed to a toxic concentration of drug and suffer unwanted side effects. Such drugs may be approved for use after small pivotal trials, but later be withdrawn after use in larger, marketed populations.
However, many mechanisms of drug action do not themselves cause toxicity. As discussed in this White Paper, we believe that GSNOR inhibition is one of them.
GSNOR regulates the nitrosylation pathways that have been in evolution for ~ a billion years. All eukaryotic organisms utilize nitrosylation for physiological control and GSNOR is the controlling enzyme. If that pathway had toxic consequences, it would have been eliminated by evolution. So, while not proof, we believe that regulating the evolutionarily conserved nitrosylation pathways by inhibiting GSNOR, would be expected, by itself, to have no toxic side effects, which, to date, the preponderance of the evidence supports.
Off-target toxicity: Occurs when a drug designed to bind to target A also binds to cellular or other targets B, C, D, etc. Such off-target binding may not be a problem if the drug-bound targets do not produce toxicity. However, if any of the targets, once bound by the drug, produce side effects, then the drug may be too toxic for use in patients and thus fail in clinical development. This problem is a drug by drug problem and must be assessed for each drug in pre-clinical and clinical development. Only those drugs that have a sufficient ratio of the Toxic dose/Therapeutic dose are allowed by FDA to be used in disease therapy. While there is no absolute limit, generally that ratio should be at least 10-fold or higher, unless the condition being treated is life-threatening and the side effects are considered acceptable to gain the therapeutic benefit. Aspirin, in dogs for example, has a ratio around 4 and might not make it through safety assessment today.
SAJE’s latest safety data shows that SPL-334.1, our first generation lead compound, has a toxic dose to therapeutic dose ratio, or therapeutic index, of > 640-fold, suggesting that the drug is quite safe. We are currently obtaining similar safety data for our second and third generation lead compounds.
Safety of GSNOR’s Mechanism of Action:
One method of assessing the likelihood of mechanistic toxicity arising from inhibiting an enzyme such as GSNOR is to eliminate or knockout, using genetic molecular biological techniques, the gene coding for that enzyme. There are normally two copies of each gene and one can knockout either one or both copies of those genes. The heterozygous knockouts with only one gene copy eliminated (+/-) are similar to the case of pharmacological inhibition of an enzyme, since drugs never totally eliminate an enzyme, but only reduce it into a therapeutically useful range. The homozygous knockouts with both gene copies missing (-/-) are more stringent, since there is none of the enzyme present from conception until death. In fact, many double knockouts (-/-) are embryonically or post-natally lethal or severally disabling, yet drugs reducing them are used therapeutically.
In terms of the GSNOR-/- mice, they have been shown to reproduce normal litters with a size and frequency similar to C57BL/6 mice. The GSNOR -/- mice developed normally and weighed the same as C57BL/6 mice. Histological examination of 4 wild-type (2 males, 2 females) and 4 GSNOR -/- mice (2 males, 2 females) showed no gross morphological or histological difference between the two mouse strains in any of the tissues studied: brain, heart, lung, liver, kidney, spleen, thymus, mesenteric lymph node, salivary gland, intestinal tract, pancreas, testis, ovary, or uterus. Blood cell counts and serum chemistries were normal in GSNOR -/- mice and their lifespan is the same as the wild-type mice ( 1 ).
Wei, et al. (2) reported that GSNOR knockout (-/-) mice started to spontaneously develop 5-20mm hepatocellular carcinoma (HCC) tumors at 1.9 years of age, which is at 88% of their lifespan (at 700 days vs 800 total), making them aged before they develop HCC. About 28% of males and 8% of females developed the tumors, compared to about 5% of wild type male and 0% of wild type females. Interestingly, no other tumor types were found to be elevated in the GSNOR knockout mice, suggesting that GSNOR knockout induced tumors are peculiar to HCC and are not a general mechanism of carcinogenesis. Wei, et al. (2) also showed that GSNOR knockout mice were more susceptible to a highly carcinogenic dose of diethylnitrosamine (DEN) that induces HCC. They found that the knockout mice had lower levels of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), which repairs the O6-ethylguanine adduct produced by DEN.
So, what implication does Wei, et al.’s work have for the development of GSNOR inhibitors? SAJE believes that GSNOR -/- mice are very different from human therapeutic uses of GSNOR inhibitors. GSNOR knockout (-/-) mice have no GSNOR expression in any tissue from conception until death, yet the animals develop, grow, behave, reproduce, and survive very similarly or identically to normal mice. Thus, complete absence of GSNOR from conception to death does not cause toxicity of the sort that extensive nitrosative stress or other toxicities would cause. Total GSNOR absence throughout life is a very different physiological condition than intermittent reduction of GSNOR as occurs after treatment with a GSNOR inhibitor. In vivo use of enzyme inhibitors typically reduces but does not eliminate enzyme activity.
In the case of the GSNOR +/- mice, the same Liu lab at UCSF (3) found that the (+/-) mice have no increase in DEN induced HCC. They concluded that AGT levels in the GSNOR (+/-) mice were the same as in wild-type mice, that the body weights of wild type and (+/-) mice were the same after DEN, that there was no increase in either tumor size or frequency in wt and (+/-) mice; and that the partial deletion or knockdown of GSNOR had no adverse sequelae. Again, the (+/-) mice are much more analogous to the clinical situation in which GSNOR activity is reduced transiently by treatment with GSNOR inhibitors, but is not eliminated. Furthermore, experts in knockout technology say to beware of conclusions about safety from knockout studies because you never know what other genes were affected by the procedure to develop the knockout animals.
In summary, the data are strong that the mechanism of inhibiting GSNOR to achieve pleotropic therapeutic activity appears to be safe with no adverse consequences.
Human Clinical Studies of GSNOR Inhibitors: Clinical Proof of Concept
SAJE has one competitor in the GSNOR inhibition field: Nivalis Therapeutics (formerly N30) in Boulder, CO. They have done multiple Phase I studies in both asthma and cystic fibrosis with two different drugs, one completed Phase II study with N6022 by iv, and a second oral Phase II study with N91115 in cystic fibrosis. The FDA just allowed orphan and fast track status for N91115 and allowed a dose escalation in the cystic fibrosis Phase II study. Furthermore, the FDA allowed them to expand the mutation pool of cystic fibrosis patients. The results of the asthma Phase II study have been published (NCT01316315) and showed anti-asthma efficacy with no serious adverse events. The cystic fibrosis Phase II study is still ongoing, but must be going well or the FDA would not have allowed dose escalation, orphan drug status, fast-track status, and enlargement of the patient pool.
All these clinical studies of two different GSNOR inhibitors suggest that there is no evidence yet to suggest that GSNOR inhibition itself presents serious safety issues. These studies constitute Clinical Proof of Concept for the GSNOR target.
structures of the Nivalis compounds are unrelated to the structures of SAJE’s compounds, so SAJE must, of course, test its compounds for off-target toxicity. SAJE finds comforting the fact that Nivalis has found clinical efficacy and no apparent clinical toxicity with either of its GSNOR inhibitors, suggesting that the mechanism of GSNOR inhibition is both safe and efficacious in asthma and cystic fibrosis.
- Limin Liu, Yun Yan, Ming Zeng, Jian Zhang, Martha A. Hanes, Gregory Ahearn, Timothy J. McMahon, Timm Dickfeld, Harvey E. Marshall, Loretta G. Que, and Jonathan S. Stamler. Essential Roles of S-Nitrosothiols in Vascular Homeostasis and Endotoxic Shock. Cell (2004), 116, 617–628.
- Wei Wei, Bin Li, Martha A. Hanes, Sanjay Kakar, Xin Chen, Limin Liu. S-Nitrosylation from GSNOR Deficiency Impairs DNA Repair and Promotes Hepatocarcinogenesis. Science Trans. Med. 2:(19): 19ra13
- See abstract: Dorothy Colagiovanni, Wei Wei, Joan Blonder, Limin Liu, and Gary J. Rosenthal. Heterozygous Deletion of S-Nitrosoglutathione Reductase in Mice Does Not Increase Nitrosative Inactivation of O6-Alkylguanine-DNA Alkyltransferase or Diethylnitrosamine-induced Hepatocarcinogenesis