key: cord-1040016-gl5cvna0 authors: Grimes, Joseph M.; Khan, Shaheer; Badeaux, Mark; Rao, Ravi M.; Rowlinson, Scott W.; Carvajal, Richard D. title: Arginine Depletion as a Therapeutic Approach for Patients with COVID-19 date: 2020-11-04 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2020.10.100 sha: d1306a5f65317d671fef2c6f014db3a1e63521d7 doc_id: 1040016 cord_uid: gl5cvna0 The COVID-19 pandemic, caused by SARS-CoV-2, is a source of significant morbidity and mortality worldwide, and effective treatments are urgently needed. Clinical trials have largely focused on direct anti-viral therapies or on immunomodulation in patients with severe manifestations of COVID-19. One therapeutic approach that remains to be clinically investigated is disruption of the host-virus relationship through amino acid restriction, a strategy utilized successfully in the setting of cancer treatment. Arginine is an amino acid that has been shown in non-clinical studies to be essential in the life cycle of many viruses. As such, arginine depletion may represent an effective therapeutic approach against SARS-CoV-2. Several arginine-metabolizing enzymes in clinical development may represent a viable approach to induce a low arginine environment to treat COVID-19 and other viruses. Herein, we explore the rationale for arginine depletion as a therapeutic approach for COVID-19. The COVID-19 pandemic, caused by the SARS-CoV-2 virus, is a tremendous burden on healthcare systems and a risk to both lives and livelihoods around the globe. Many clinical trials are underway, with current strategies focusing on directly disrupting viral machinery, such as with the RNA-polymerase inhibitor remdesivir, or modulating the intensity of the immune response, such as with the anti-cytokine antibodies tocilizumab and sarilumab. Manipulation of the hostvirus relationship to disrupt the viral lifecycle represents a novel therapeutic strategy. Like other viruses, SARS-CoV-2 is obligately reliant upon host machinery and nutrients for the synthesis of viral macromolecules. The deprivation of key nutrients-an approach used in the oncology field to treat tumors-may therefore interfere with viral replication. Although this metabolic starvation approach has yet to be clinically applied to virus control, preclinical studies support this concept. Arginine is a key nutrient shown to be essential in vitro in the lifecycle of many DNA and RNA viruses, and therapeutic depletion of arginine may therefore inhibit SARS-CoV-2 replication. Several arginine-depleting enzymes are already in clinical development (Supplemental Table 1 ). Pegzilarginase is an engineered, pegylated enzyme that degrades arginine to produce ornithine and urea and is currently in a phase 3 clinical study in patients with Arginase 1 deficiency. PEGylated recombinant human arginase 1 (BCT-100) is currently in development to treat malignancies. Pegylated arginine deiminase (ADI-PEG 20) is a bacterial enzyme that employs a different enzymatic mechanism, generating citrulline and ammonia from arginine. In a recent cancerfocused clinical trial, ADI-PEG 20 was shown to have antiviral activity in HCV patients with hepatocellular carcinoma. These arginine-degrading therapeutics may therefore represent readily J o u r n a l P r e -p r o o f accessible treatments for COVID-19. Herein, we review the evidence for arginine depletion as a strategy to treat SARS-CoV-2 infection. Arginine is a semi-essential amino acid that can be obtained from the diet or produced within certain cells via the complete or partial urea cycle. In addition to its important role in the makeup of essential proteins, arginine is a substrate for the various isoforms of nitric oxide synthase (NOS), which converts arginine into nitric oxide (NO) and citrulline. Both citrulline and ornithine, as direct products of arginine metabolism, also undergo further modifications into other bioactive compounds. 1 Within the urea cycle, arginine is converted by arginase 1 (ARG1) into ornithine and urea, thereby allowing excretion of excess nitrogen produced by protein catabolism. Ornithine is then recycled to re-make arginine within the urea cycle through use of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthetase 1 (ASS1), and arginosuccinate lyase (ASL) (Figure 1 ). Of these urea cycle enzymes, only the liver and parts of the small intestine express all four, as most other cells in the body do not express OTC. The importance of this expression pattern and of the expression level of ASS1 as pertaining to arginine deprivation as an antiviral therapeutic approach will be discussed below. In an arginine-deprived environment, arginine is generated endogenously from either ornithine (for those cells that express OTC), or citrulline (for cells that express ASS1 and ASL but not OTC). Therefore, in the context of treatment with the ornithine-generating enzymes Pegzilarginase or J o u r n a l P r e -p r o o f BCT-100, most cells in the body will not be able to endogenously synthesize arginine (Figure 1 ). To compensate for the low arginine environment, cells up-regulate ASS1 and convert circulating citrulline into arginine, consuming aspartic acid in the process. 2 The importance of aspartic acid availability for viral replication was highlighted by Grady et al. as they showed that a low ASS1 expression status, which would allow for continued use of aspartic acid as a substrate for pyrimidine synthesis rather than for arginine production, favored replication of the herpes simplex viruses (HSV). 3 This same concept has also been proposed as a rationale for the relatively faster proliferation rates of ASS1 deficient tumor cells. 4 Based on this link between arginine synthesis via the urea cycle and pyrimidine production, it is interesting to contemplate the potential additive or synergistic effects of combining an arginine depleting approach with nucleotide analog therapies such as remdesivir. Arginine depletion has long been investigated in vitro as a potential antiviral strategy with the majority of studies performed on the Herpesviridae and Adenoviridae families. A number of other RNA and DNA viruses have also been investigated (Supplemental Table 2 ), but, to date, no studies of arginine depletion in the coronavirus family have been found. Early studies show that HSV viral yield improves with increasing arginine levels in the medium of HSV-1 infected cells and that complete arginine deprivation inhibits viral replication. 5 6 Viral DNA synthesis was not affected by acute arginine depletion, which suggests that either viral RNA synthesis or protein production impact viral replication. 6 Of note, re-introduction of arginine after deprivation in HSV-1 infected cells resulted in resumption J o u r n a l P r e -p r o o f of normal virus production thereby highlighting the importance of arginine. 5 Given these early findings, it was postulated that arginase activity may be a key anti-viral tool. A supporting experiment found that release of arginase by macrophages inhibits viral replication of HSV-1, implying that control of local arginine levels is an anti-viral mechanism. 7 More recently, arginine depletion was studied in HSV-1-infected cells treated with a pegylated form of native arginase 1 (Peg-ArgI). Peg-ArgI treatment inhibits viral replication, halts production of viral progeny with reduction in cell-to-cell transmission, and blocks the classic cytopathic effects of HSV-1. Furthermore, Peg-ArgI exhibited more anti-viral activity compared to acyclovir. 8 Cytomegalovirus (CMV) has also been shown to replicate in a dose-dependent manner as arginine is introduced to cell culture medium. 9 Contrary to findings in the HSV studies, the anti-viral effects of arginine deprivation impacted DNA production. Another study in a murine CMV model noted that arginine deprivation decreased viral DNA, RNA, and protein production, but could not conclude that synthesis of these macromolecules was completely inhibited. 10 Both of these studies demonstrated that re-introduction of arginine stimulates viral production up to 8 days later. Adenovirus. Early studies of amino acid deprivation in adenovirus identified arginine as an essential nutrient in viral replication. 11 Interestingly, these studies found that viral DNA and protein accumulate even in the absence of arginine. 12 13 However, upon re-introduction of arginine, these viral components cannot be packaged into mature virions, and that viral lifecycle depends on synthesis of new components. 14 This suggests that viral proteins made in the absence of arginine J o u r n a l P r e -p r o o f are defective or inaccessible to virion packaging. One study also notes that packaging of viral DNA into new capsids may require an arginine-dependent process. 13 Experience with Other Viruses. Other viruses have been studied in the context of arginine deprivation ( Supplemental Table 2 ). The RNA influenza virus has conserved arginine residues critical for viral replication, which is in line with previous studies showing decreased influenza viral yield in cultures depleted of arginine. 15 16 The vaccinia virus is a DNA virus shown to be dependent on arginine for both early (DNA production) and late (virion packaging) viral stages. 17 Arginine depletion studies in the polyomavirus Simian Virus 40 (SV40) show that DNA and viral proteins can be synthesized but not packaged into mature virions, similar to findings in the adenovirus. 18 19 Measles Morbillivirus, the RNA virus responsible for measles, is not essentially dependent on arginine, but low arginine levels reduce viral progeny. 20 The DNA alphaherpes virus responsible for Marek's disease in poultry is reliant on arginine for viral protein formation, but not for viral entry or DNA synthesis. 21 In support of the concept and safety of therapeutic arginine depletion as an anti-viral approach is a retrospective analysis of a clinical study of hepatocellular carcinoma (HCC) patients treated with ADI-PEG 20. 22 This study demonstrated that 5 out of 10 HCV serotype 1B patients had a greater than 90% reduction in viral load. Decreases of 47-82% were demonstrated in 3 other patients. These data are striking given the potential rescue effect associated with the generation of citrulline, as well as the high incidence of anti-drug antibody formation. 23 It is possible that the latter may have contributed to the variability in the HCV titer response. In addition to the putative direct anti-viral activity in SARS-COV-2, arginine depletion may also attenuate the pulmonary inflammation seen in COVID-19 infection. Severe cases of COVID-19 are characterized by a hyperinflammatory lung pathology and local tissue damage that contribute to poor patient outcome. 28 Arginine is the substrate for NOS-mediated production of NO, a signaling molecule that is a key mediator of the innate inflammatory immune response imparted by viral infections. 29 In experimental models of severe influenza infection, which shares in common with COVID-19 a state of pulmonary hyperinflammation, induced NO overproduction directly contributed to animal morbidity and mortality. 30 Clinically, reduction of NO has been demonstrated following chronic dosing with ADI-PEG 20 in HCC/HCV patients. 22 By limiting the availability of arginine for production of NO, especially production of NO mediated by iNOS/NOS2, it may be possible to limit the extent of the hyperinflammatory response in COVID- Pegzilarginase. Pegzilarginase has been studied in a number of clinical trials in patients with solid (NCT02561234) and hematological malignancies (NCT02732184), and is currently in Phase 3 clinical development for the treatment of patients with Arginase 1 deficiency (NCT03921541). Pegzilarginase is well-tolerated and shows sustained reduction of serum arginine concentration to  10% of normal-range baseline levels for ≥48 hrs. BCT-100. BCT-100 has primarily been studied as an anti-neoplastic agent as a single agent (NCT01092091) and in combination with other therapies (NCT02089633). BCT-100 is generally well-tolerated and demonstrates sustained reduction in serum arginine levels in weekly repeat dosing trials. Anti-BCT-100 antibodies have not been detected in patients. 33 All clinical trials involving arginine-depleting enzymes can be found in Supplemental Table 3 . Although arginine depletion has long been proposed as a potential anti-viral mechanism, this strategy has yet to be applied in the clinic. Given the preclinical evidence summarized in this paper, arginine appears to be a key metabolite important for successful viral replication, and there are clear steps in the SARS-CoV-2 viral lifecycle that rely on conserved arginine residues . intestines as OTC is not expressed (grey); therefore, arginine cannot be synthesized and utilized for viral replication. In contrast, citrulline produced by ADI-PEG 20 is more easily converted to arginine outside of the liver since ASS1 and ASL are expressed in most tissues; therefore, viral replication may not be impeded as effectively. A consequence of ASS1 upregulation is that any available citrulline will be rapidly conjugated to aspartate, preventing its utilization in pyrimidine ring synthesis thereby restricting viral replication of another key building block. OTC=Ornithine Arginine deprivation as a targeted therapy for cancer Argininosuccinate synthetase 1 depletion produces a metabolic state conducive to herpes simplex virus 1 infection Diversion of aspartate in ASS1-deficient tumours fosters de novo pyrimidine synthesis Requirement of Arginine for the Replication of Herpes Virus The Role of Arginine in the Replication of Herpes Simplex Virus Inhibition of herpes simplex virus multiplication by activated macrophages: a role for arginase? Development and evaluation of a host-targeted antiviral that abrogates herpes simplex virus replication through modulation of arginine-associated metabolic pathways The effect of arginine deprivation on the cytopathogenic effect and replication of human cytomegalovirus Effect of Arginine Deprivation on Murine Cytomegalovirus 11. Rouse HC, Schlesinger RW. An arginine-dependent step in the maturation of type 2 adenovirus Mechanism of the arginine requirement for adenovirus synthesis. I. 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