Repositioning PARP inhibitors for SARS-CoV-2 infection (COVID-19); a new multi pronged therapy for ARDS?

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Salient features:

This article focuses on:

  • Characteristics of the SARS-CoV-2 (COVID-19) infection
  • Brief introduction to the biology of PARPs
  • Therapeutic potential of PARP inhibitors
  • Anti-inflammatory effects of PARP inhibitors, modulation of cell death – combatting the cytokine storm
  • Ventilator-induced lung injury
  • Antiviral effects of PARPs and its interactions with coronaviruses
  • Interactions with drugs proposed for COVID-19 therapy
  • Available PARP inhibitors and their current scope of use
  • Concluding remarks

Detailed summary:

Characteristics of the SARS-CoV-2 (COVID-19) infection

  • The newly emerging coronavirus disease, COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
  • The SARS-CoV-2 belongs to the Coronaviridae family, being genetically related to the human pathogen SARS-CoV-1 and MERS-CoV and to a number of bat-origin coronaviruses.
  • SARS-CoV-2 is an enveloped virus that has a positive sense single stranded RNA (ssRNA+) genome of nearly 30,000 nucleotides in length.
  • The first site of viral infection is the upper respiratory tract.
  • At a later stage of infection the virus may disseminate to and replicate in the lower respiratory tract.
  • Viral RNA and infectious virus can be found in the nasopharyngeal swab and the sputum.
  • Infection of the gastrointestinal tract has been reported and infectious viruses can be isolated from fecal specimens.
  • The virus uses the Angiotensin-converting enzyme 2 (ACE2) as cellular receptor and a type II transmembrane serine protease, TMPRSS2, as co-factor that activates the attachment protein of SARS-CoV-2 to aid viral entry into epithelial cells of the respiratory and in the gastrointestinal tract.
  • In the majority of the cases, patients have very mild symptoms.
  • A considerable portion of the patients develop severe symptoms and require intensive care and mechanical ventilation; this patient group shows an increased risk for death.
  • The risk factors for complications and mortality include pre-existing cardiovascular, metabolic or neoplastic diseases and older age.
  • Men have a higher chance for developing severe symptoms, as well as, for fatal outcomes.
  • A detrimental sequel of SARS-CoV-2 virus infection is cytokine storm that occurs after the elimination of the virus and culminates in multiple organ failure.
  • Cytokine storm may be responsible for a considerable fraction of adverse outcomes in SARS-CoV-2 infection.
  • In clinical practice Tocilizumab, an antibody against IL-6 and Anakinra, an IL-1 receptor antagonist were used successfully to cope with SARS-Cov-2-induced cytokine storm suggesting the involvement of these interleukins.
  • Other drugs, primarily used in rheumatologic settings, are also suggested to be used to block cytokine storm.

Brief introduction to the biology of PARPs

  • Poly(ADP-ribose) polymerase (PARPs/ARTDs) enzymes constitute a family of 17 members in humans (PARP1-PARP17).
  • When activated, PARPs cleave their substrate, NAD+, and couple the resulting ADP-ribose units onto acceptor proteins forming mono, oligo or polymers of ADP-ribose.
  • The polymer of ADP-ribose is called poly(ADP-ribose) (PAR).
  • The large, negatively charged mono, oligo or polymers heavily impact on the behavior of target proteins and a huge number of proteins were shown to be ADP-ribosylated or PARylated on multiple different amino acids.
  • ADPribose units can serve as binding surfaces for proteins.
  • Macrodomain-containing proteins constitute a small protein family.
  • Macrodomains can bind to ADP-ribose units and some can hydrolyze ADP-ribose units upon binding.
  • Through binding or hydrolyzing ADP-ribose, macrodomain containing proteins can translate ADP-ribosylation signals into cellular adaptation programs.
  • Macrodomains can be found in all domains of life, including viruses and more specifically, coronaviruses.
  • PARP1 is responsible for ~85-90% of cellular PARP activity, PARP2 is responsible for 10-15%, while the rest of the enzymes share the remainder of all cellular PARP activity.
  • Active PARPs represent a large burden on cellular NAD+ levels.
  • PARP activity under physiological conditions also represents a large burden on cellular NAD+ levels.
  • PARP1, PARP2 and PARP3 can be activated by binding to irregular or damaged DNA that is often the result of reactive oxygen (ROS) or reactive nitrogen species (RNS) production under inflammatory conditions.
  • The activation of PARP1, PARP2 and PARP3 is vital for initiating DNA repair and the resolution of irregular DNA structures and modulating chromatin structure.
  • Through these, certain PARP isoforms (chiefly, PARP1) are involved in recombination and transcription events that encompass changes to DNA structure.
  • PARP2 can bind to RNA, which may activate the enzyme.
  • There are other pathways to activate PARPs involving signal transduction pathways.
  • PARP enzymes were shown to be involved in transcription, mRNA handling and polypeptide elongation.
  • PARP enzymes are involved in a wide variety of cellular processes, among these, their impact in cell death, immune function, antiviral response, transcription, translation and autophagy are relevant.
  • PARP inhibitors (PARPi) that are in current clinical use (Olaparib, Rucaparib, Niraparib and Talazoparib) are inhibitors of PARP1, PARP2 and PARP3.

Therapeutic potential of PARP inhibitors

Anti-inflammatory effects of PARP inhibitors, modulation of cell death – combatting the cytokine storm

  • The protective effect of PARPi, that is applicable to non-oncological models of inflammation, was first suggested by Nathan Berger’s group nearly 40 years ago.
  • They noted that DNA damage-induced cell death was associated with PARP activation, resulting in massive depletion of its substrate, NAD+, and consequently ATP that resulted in necrosis.
  • Depletion of these pools was prevented in the presence of a PARPi and necrosis reduced.
  • Subsequently it has also been shown that the product of the PARP reaction, i.e. PAR can also trigger apoptosis-inducing factor (AIF) release that promotes a specific programmed cell death pathway called “parthanatos”.
  • Oxidative and nitrosative stress, which is integral to inflammation causes DNA strand breakage that massively activates PARP.
  • PARP can actively increase and prolong inflammation through a vicious cycle of ROS/RNS-induced DNA damage, PARP-mediated necrosis and increase in inflammatory cytokines.
  • PARPi have been shown to limit inflammation-induced normal tissue damage, including acute lung injury, in animal models.
  • Reactive species production increases in SARS-CoV-2 (COVID-19) infection that makes PARP activation and PARP activation-mediated cell death likely.
  • Pharmacological PARP inhibition is likely to reduce cell death under these conditions as suggested by the study on another ssRNA(+) virus, the Zika virus. Zika virus-induced cell death was effectively blocked by PARP inhibition.
  • The source of the reactive species can be diverse, stemming from activated immune (e.g. macrophages) or dysfunctional cells.
  • The nucleocapsid protein of SARS-CoV-1 can induce reactive oxygen species production in non-immune cells too that can be a pathway in the case of SARS-Cov-2 as well.
  • PARP1 is the best characterized member of the PARP family from the perspective of immune processes. PARP1 seems to have mostly pro-inflammatory properties in terms of Th1 and Th2-mediated processes.
  • PARP2 has limited role in pro-inflammatory processes.
  • Pharmacological PARP inhibition is generally anti-inflammatory.
  • PARP1 is involved in regulating innate and adaptive immunity through mediating signal transduction and transcription events in multiple immune cell lines that is translated to differential expression of cytokines and chemokines, as well as, their receptors.
  • In humans there is evidence that the polymorphism in PARP1 (V762A) that confers reduced activity, is associated with reduced risk of asthma.
  • PARP activity is increased in the lung tissues of asthmatic patients and in mouse models the PARPi, olaparib, prevents asthma indicating that the extrapolations from preclinical models to human conditions are valid.
  • PARP inhibition confers protection to the lungs upon acute lung injury inflicted by various noxae (burn, smoke inhalation, bacterial infection, etc.) in a nuclear factor-κB (NFκB)- dependent fashion.
  • Various PARPi (3-AB, PJ-34 and INO-1001) have been shown to be protective in various preclinical models of acute respiratory distress syndrome (ARDS) by reducing production of inflammatory mediators and preventing depletion of NAD+ and ATP, and also the deterioration of barrier function that may contribute to exudate formation in ARDS.
  • The most common animal model of human acute lung injury/ARDS is intratracheal instillation of lipopolysaccharide (LPS).
  • PARP plays an important role in the pathogenesis of LPS-mediated ARDS damage by a variety of mechanisms, and both genetic disruption of PARP1 and inhibition of PARP ameliorates ARDS and ARDS-associated tissue damage.
  • Following LPS administration olaparib not only reduced inflammatory cell infiltration in the lung and pulmonary oedema and exudate, but also reduced secondary kidney injury.
  • Relevance to human lung disease comes from the observation that in cases of chronic obstructive pulmonary disease (COPD), plasma NAD+ is low and both DNA damage, PARP activity and the percentage of PAR positive lymphocytes were higher than in control subjects.
  • PARP inhibition has an additional lung protective feature. PARPi’s are able to reduce lung fibrosis, a common sequel of SARS-Cov-2 lung inflammation, in different preclinical animal models.
  • PARP activity in idiopathic lung fibrosis patients was higher than in healthy controls suggesting that the findings of the animal models can be translated to the human situation.
  • A major reason of mortality in SARS-CoV-2 (COVID-19) infection is the macrophage overactivation that leads to cytokine storm and, consequently, to multi-organ failure
  • To date, IL-6 and IL-1 were implicated in SARS-CoV-2 (COVID-19)- induced cytokine storm, however, the involvement of tumor necrosis factor α (TNFα) is also likely.
  • PARPi’s, including olaparib can reduce the expression of IL-6 in multiple organs, including the lung in animal models.
  • Pharmacological PARP inhibition using INO-1001 in humans reduced serum interleukin-6 (IL-6) and C-reactive protein (CRP) levels further reinforcing the notion that data from preclinical studies can be translated to humans.
  • Another interleukin implicated in eliciting the cytokine storm in SARS-CoV-2 (COVID-19) infection is interleukin-1 (IL-1).
  • PARPi’s, including olaparib, can decrease IL-1β expression in animal models that, again, points out the applicability of PARPi’s to dampen IL-1 expression in humans. Finally, pharmacological PARP inhibition, including with the clinically approved PARPi, olaparib, reduced TNFα levels in the lungs in various animal models of lung inflammation.
  • PARP inhibition can counteract inflammationinduced cell death, possibly counteracts the ARDS-like features and cytokine storm through blocking macrophage overactivation (cytokine storm) through downregulating the expression of IL-6, IL-1 and TNFα in humans. These events represent a vicious circle that is sustained by PARP1 activation, therefore, PARPi can break the chain of events at multiple loci and exert cytoprotective effects on the pulmonary epithelial and endothelial cells.

Ventilator-induced lung injury

  • SARS-Cov-2-infected patients requiring intensive care often need mechanical ventilation over extended periods.
  • Pharmacological inhibition of PARP reduced the inflammatory component of ventilation-induced lung damage in a murine model.
  • In case of severe acute lung injury the PARPi PJ34 protects the kidney from injury following mechanical ventilation.
  • Similar findings were obtained in an ovine model where acute lung injury was induced by smoke inhalation and local Pseudomonas aeruginosa colonization.
  • It appears that in ventilation-induced lung damage (VILI) PARP inhibition breaks a similar vicious cycle as in ARDS or other inflammatory models.

Antiviral effects of PARPs and its interactions with coronaviruses

  • Several members of the PARP family are involved in host-virus interactions, most PARP enzymes have antiviral properties.
  • PARPs impact on multiple steps of the viral life cycle, these steps are mostly related to the nucleic acid-binding properties of PARPs.
  • PARPs can interfere with viral integration, recombination and transcription, however, these are not relevant in the context of coronaviruses. PARP13 was shown to be induced upon orthomyxovirus or alphavirus infection and to have protective function.
  • Interferon treatment induces a set of PARP enzymes PARP9, PARP10, PARP12 and PARP14 in cells.
  • These PARP enzymes inhibit viral translation probably through ADP-ribosylating key cellular proteins.
  • Members of the virus families Coronaviridae, Togaviridae and Hepeviridae encode macrodomain proteins that bind to and hydrolyze ADP-ribose from proteins and are critical for optimal replication and virulence.
  • The conserved coronavirus macrodomain in SARS-CoV was found to play a role in viral replication and to suppress IFN and cytokine production
  • The nucleocapsid protein of SARS-CoV and a number of other CoV’s are ADP ribosylated.
  • Since PARP inhibitors have preference towards PARP1, PARP2 and PARP3 it is unlikely that PARPi treatment would interfere with the antiviral effects of the minor PARP isoforms.
  • Another PARPi, PJ34, can form a complex with the nucleocpasid protein of the human coronavirus CoV-OC43 and, hence, can hinder its RNA binding affinity.

Interactions with drugs proposed for COVID-19 therapy

  • Combination therapy is advantageous, since it requires lower doses with better tolerability of the components.
  • PARPi’s have not been reported to interact with Ritonavir, Lopinavir or Remedsivir.
  • Interferon beta, has pleiotropic antiviral actions, and therefore, probably interferon beta can induce the antiviral function of PARPs.
  • Chloroquine and hydroxychloroquine are antimalarial and antirheumatic drugs that were assessed in small scale cohorts in France and China.
  • Chloroquine and hydroxychloroquine probably block the processing of SARS-CoV-2 by blocking the acidification of the vesicles containing the virus, in a process biochemically similar to the process of autophagy.
  • The genetic or pharmacological inhibition of PARP1 blocks the early steps of autophagy.
  • Furthermore, the genetic or pharmacological inhibition of PARP2 blocks the degradation of the cargo of autophagic vesicles mimicking the action of chloroquine.
  • PARPi’s could potentiate the antiviral effects of chloroquine or hydoxychloroquine, and could be exploited to achieve dose reduction of chloroquine or hydoxychloroquine.
  • ADP-ribosylation-mediated autophagic processes were found to be important in the pathogenesis of other microorganisms such as Legionella in high-profile studies.
  • IL-6 is a key interleukin in sustaining cytokine storm following SARS-CoV-2 infection.
  • Studies have shown that anti-IL-6 immunotherapy using Tocilizumab was beneficial for patients.
  • PARP inhibition was shown to reduce IL-6 expression in humans, therefore, it is possible that PARP inhibition could also potentiate the effects of anti-IL-6 (Tocilizumab, Siltuximab) or antiIL-6 receptor (Sarilumab) treatment.
  • PARPi’s can reduce IL-1 and TNFα expression it is possible therefore, that PARPi’s could potentiate Canakinumab (IL-1β antibody), Anakinra (IL-1 receptor antagonist) and Adalimumab (TNFα antibody) therapy.

Available PARP inhibitors and their current scope of use

  • Four PARPi are approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for cancer therapy.
  • Although, the original rationale for their development was to overcome DNA repair-mediated resistance to DNA damaging anticancer therapy, their approval has been as single agents exploiting tumor-specific defects in the complementary DNA repair pathway homologous recombination repair (HRR) by a process known as synthetic lethality.
  • PARPi are only approved for cancer therapy as single agents.
  • The first to be approved was olaparib, now called Lynparza®.
  • Originally approved by the FDA at the end of 2014 at a dose of 400 mg twice daily in capsule formulation, it is now approved in tablet formulation at 300 mg twice daily for maintenance (including front-line) therapy in ovarian cancer.
  • It is also approved at the same dose and schedule for metastatic breast and pancreatic cancer in individuals with germline BRCA mutations.
  • Also approved in ovarian cancer are rucaparib (Rubraca®) at 600 mg orally twice daily and niraparib (Zejula®) 300 mg orally once daily.
  • Talazoparib (Talzenna®) is the most potent of all the PARPi and has been approved at dose of only 1 mg daily for germline BRCA mutated metastatic breast cancer.
  • Veliparib is a less potent PARPi that has so far failed to show sufficient single agent activity for approval but has been in advanced clinical trial alone and in combination with other anticancer agents for over a decade.
  • Due to their tumor-specific synthetic lethality their toxicity as monotherapies is mild and manageable.
  • The common adverse effects are fatigue, gastrointestinal (nausea/vomiting, abdominal pain, diarrhea) and haematological (neutropenia for olaparib, anaemia for rucaparib and talazoparib, thrombocytopenia for niraparib), mostly grade 1-2.
  • Myelodysplastic syndrome and acute myeloid leukemia have also been noted, but this may have been caused mainly by prior chemotherapy, particularly platinum-based therapies.
  • In combination with DNA damaging therapies, the safe dose is much lower and the tolerated schedules are much shorter. For example, the approved dose of rucaparib (Rubraca®) is 600 mg orally 2x daily continuously, the safe dose in combination with carboplatin is 240 mg once daily for 14 days every 21 days and only 18 mg/m2 i.v [equivalent to 60 mg orally] for 5 days every 28 days in combination with temozolomide.
  • It is anticipated that the doses needed for non-oncological diseases e.g. ARDS is likely to be lower (and hence less toxic) for 2 reasons.
  • Firstly, the doses found to be effective preclinically in non-oncological models are 1-2 orders of magnitude lower than the monotherapy doses used in cancer models.
  • This is likely to reflect the intracellular concentrations needed to maintain NAD+ pool from excessive depletion vs. those needed to completely block repair for a sustained period.
  • Secondly, the approved doses are largely based on dose escalation Phase I trials where the end-point is tolerability, which may not be appropriate for a tumor-specific drug and may be well in excess of the effective dose.

Concluding remarks

  • PARPi’s may have beneficial effects in SARS-CoV-2 infection and its sequelae by preventing macrophage overactivation and the subsequent cytokine storm, as well as, by protecting cells against cell death.
  • PARP inhibitors were protective against risk factors for bad clinical outcomes of SARSCoV-2 infection, such as cardiovascular and metabolic diseases.
  • PARPi’s are cytoprotective in the central nerve system and the cardiovascular system, the systems that are damaged in COVID-19 patients with bad clinical outcome.
  • Of particular interest regarding COVID-19 is the sex bias of the mortality statistics. Men are more than twice as likely to die as women.
  • Previous research has shown that men have on average 40% higher PARP activity than women, at least in their PBMCs, with similar sex differences in mice.
  • In those studies where both sexes were included, the protective effects of PARPi were less pronounced in females.
  • There are no data for sex differences in PARP activity in human lung tissue due to the difficulty of obtaining viable normal lung tissue.
  • In humans following traumatic brain injury men were 2.6 times more likely than women to have elevated PAR-modified proteins in their CSF after comparable levels of trauma and, strikingly similar to the studies in PBMCs, the mean PAR level was approximately 40% higher in males than females.
  • The preclinical evidence suggests that 5-10% of the currently approved PARPi doses would be sufficient/effective and tolerable.

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