Introduction
The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been
identified as the disease-causing pathogen of Coronavirus disease 2019 (COVID-19).
(Pre)clinical research to identify rapidly available small molecules for the treatment
of SARS-CoV-2 infections/COVID-19 has focused to date on the approved lysosomotropic
antimalarials chloroquine and hydroxychloroquine, the investigational remdesivir (GS-5734,
compassionate use), and the anti-inflammatory corticosteroid dexamethasone (COVID-19
Treatment Guidelines Panel, 2020). Lopinavir/ritonavir and other HIV protease inhibitors,
however, were discontinued as treatment options in COVID-19 demonstrating no clinical
benefit in clinical trials.
Despite encouraging results in treating hospitalized patients with COVID-19 requiring
supplemental oxygen, mechanical ventilation, or extracorporeal membrane oxygenation
(ECMO) with remdesivir and dexamethasone, there is still a lack of active compounds
exhibiting pan-coronavirus antiviral activity, tackling or preventing host cell infection,
forming syncytia, endotheliitis, or the cytokine release syndrome (CRS)/cytokine storm
syndrome in COVID-19. Target-oriented and in particular site of action-oriented drug
repurposing of small molecules has the potential to close the gap in prophylaxis and
treatment of mild and moderate COVID-19 and to reduce mortality in severe cases.
Oxidative Stress, Apoptosis, Multinucleate Syncytia, Host Cell Entry, and Cytokine
Storm Syndrome Define Drug Repurposing Targets
Oxidative stress (e.g., enhanced ROS levels) has been demonstrated in animal models
of SARS (Delgado-Roche and Mesta, 2020) and serves as a possible explanation why SARS-CoV-2
patients with Glucose-6-phosphate dehydrogenase (G6PD) deficiency develop intravascular
hemolysis and methemoglobinemia (Palmer et al., 2020). Both, chloroquine and hydroxychloroquine,
are supposed to trigger severe drug-induced hemolytic anemia in G6PD-deficient COVID-19
patients (Beauverd et al., 2020; Kuipers et al., 2020).
Severe COVID-19 is associated with an atypical diffuse alveolar damage, ending in
the acute respiratory distress syndrome (ARDS) (Huang et al., 2020), most likely accompanied
by occurrence of syncytia as a result of a direct infection of cells by an infected
neighboring cell without releasing a complete virus (Ou et al., 2020).
Ceramides, in particular C18-ceramide, are present in (sepsis-induced) cardiac dysfunction
(Chung et al., 2017), and are effective in triggering exocytosis in rat PC12 cells
(Tang et al., 2007); further they may contribute to SARS-CoV-2-related cell–cell fusion
by exocytosis of viral S protein fractions and development of multinucleate syncytia.
Non-structural protein nsp2 of SARS-CoV-2 was associated with host cell cell cycle
progression, and apoptosis in host cells, suggesting an impact on disrupting the host
cell environment (Yoshimoto, 2020) and apoptosis of endothelial cells (Varga et al.,
2020).
According to current knowledge, cleavage-mediated fusion of viral S protein with host
cells can occur either immediately at the cell surface by TMPRSS2 or within the lysosome
catalyzed by lysosomal cathepsin L (Belouzard et al., 2012). The lysosomal cathepsin
L induced fusion of SARS particles bound to ACE2 with host cells (Millet and Whittaker,
2015) is sensitive to lysosomal pH. Hence both, TMPRSS2 and cathepsin L, display promising
targets of prophylaxis and treatment of SARS-CoV-2 infection/COVID-19.
In severe COVID-19, SARS-CoV-2 is likely to cause both, pulmonary and systemic inflammation,
thus leading to multi-organ dysfunction in high risk populations. Significantly higher
concentrations of IL-8, TNFα, and IL-6 in deceased patients (Chen et al., 2020) are
suggesting a rapid and severe deterioration during SARS-CoV-2 infection associated
with CRS/cytokine storm syndrome (Mehta et al., 2020).
Lysosomotropic (Active) Compounds Are Valuable Drug Candidates
Lysosomotropism is a biological characteristic of small molecules and always present
in addition to intrinsic pharmacological effects. Various well-known approved drugs
such as amitriptyline, chlorpromazine, sertraline, and imipramine share lysosomotropic
characteristics (Figure 1A) (Kornhuber et al., 2008; Blaess et al., 2018). Regardless
of their pharmacological effects, they are accumulating in lysosomes raising the lysosomal
pH from 4.5–5 to 6–6.5, beyond the optimum of most of the lysosomal enzymes, including
cathepsin L. Since no effects of lysosomotropic aminoglycoside antibiotics on free
cathepsin L (Zhou et al., 2016) or other lysosomotropic drugs on lysosomal enzymes
such as acid sphingomyelinase exist (Blaess et al., 2018), a selective inhibition
is unlikely.
FIGURE 1
(A) Variety of approved lysosomotropic compounds for various indications (Kornhuber
et al., 2008; Blaess et al., 2018). Achievement of the desired lysosomotropic effect
depends on the active compound, the dosage, and accumulation in lysosomes. Unless
indicated, maximum daily doses are split into three applications. *Lysosomotropism
very likely, but not yet confirmed, lysosomal drug concentration (effect) within the
therapeutic margin expected; dosage: #single dose per day; x
in vitro anti-SARS‐CoV tested, xx
in vitro anti-SARS-CoV and anti-SARS-CoV-2 tested (Vincent et al., 2005; Kornhuber
et al., 2008; Dyall et al., 2014; Zhou et al., 2016; Blaess et al., 2018; Liu et al.,
2020; Weston et al., 2020). (B) Cellular targets, cellular effects, and effects related
effects of lysosomotropic active compounds in SARS-CoV-2 infection/COVID-19 (Vincent
et al., 2005; Masters, 2006; Mingo et al., 2015; Zhou et al., 2016; Blaess et al.,
2018; Varga et al., 2020; Zhou et al., 2020). Lysosomotropic compounds target in mammalian
cells three major targets related to SARS-CoV-2 infection/COVID-19: cathepsin L (1),
gene expression of inflammation-relevant genes (2), C16-ceramide and C18-ceramide
synthesis, and apoptosis of host cells (3). Addressing targets 1–3 results in various
disease process interfering effects supposed to improve SARS-CoV-2 infection/COVID-19
outcome; (°) in viral infection and bacterial superinfection, (°°) only in bacterial
superinfection.
Lysosomotropic compounds are not limited to mediate inactivation of cathepsin L Figure
1B. Moreover, lysosomotropic compounds are assumed to suppress the CRS/cytokine storm
syndrome and to attenuate the transition from mild to severe SARS-CoV-2 infection/COVID-19
(Zhou et al., 2020). Data of the lysosomotropic model compound NB 06 in LPS-induced
inflammation in monocytic cells (Blaess et al., 2018) supports the hypothesis. NB
06 affects gene expression of the prominent inflammatory messengers IL1B, IL23A, CCL4,
CCL20, and IL6; likewise, it has beneficial effects in (systemic) infections involving
bacterial endotoxins by targeting the TLR4 receptor pathway in sepsis. Similarly,
desipramine reduces endothelial stress response in systemic inflammation (Chung et
al., 2017).
Apoptosis of (infected) mammalian cells is characterized by an increase in C16-ceramide
(Thomas et al., 1999) and can be blocked via lysosomotropic compounds such as NB 06,
chlorpromazine, and imipramine (Blaess et al., 2018). Furthermore, C18-ceramide triggered
exocytosis and forming of syncytia is blocked by chlorpromazine as well (Garner et
al., 2010).
Suitable Drug Profiles and Routes of Administration
According to current knowledge, in therapy inhibition of lysosomal pH dependent processes
(e.g., cathepsin L dependent viral entry into host cells) can be obtained only through
off-label use of lysosomotropic drugs. Systemic application in lysosomotropic drug
concentrations and obtaining an efficacious blood level is sometimes accompanied by
severe adverse effects and/or (in this case) undesirable (intrinsic) pharmacological
effects. Chloroquine was among the first lysosomotropic active compounds exerting
antiviral effects on SARS-CoV-2 (Liu et al., 2020) and during SARS-CoV pre- and post-infection
conditions (Vincent et al., 2005). Owing to an unfavorable drug profile (G6PD patients,
insufficient lysosomotropism, elimination half-life of 45 ± 15 days), a recommendation
against (hydroxy)chloroquine, but not against lysosomotropic active compounds in principle
was issued (COVID-19 Treatment Guidelines Panel, 2020).
Chlorpromazine displayed anti-SARS-CoV-2 effects in vitro (Weston et al., 2020) and
protective effects on COVID-19 in patients in a psychiatry hospital (NCT04366739).
Consequently, chlorpromazine is rated as a promising candidate in COVID-19/CRS treatment.
In case of treatment of people without mental illness, however, a premature termination
of treatment due to severe side effects by systemic application of chlorpromazine
is extremely likely. This raises the question of how to handle this issue to provide
well tolerated lysosomotropic drugs in SARS-CoV-2 infection/COVID-19.
Personalized Bench to Bedside Treatment Concept
Numerous available approved drugs with lysosomotropic characteristics permit tailor-made
therapy. The individual pre-existing conditions are a criterion for the selection
and combination of lysosomotropic drugs. For choosing suitable lysosomotropic drugs
some issues have to be considered:
Tolerable Intrinsic Pharmacology and Drug Profile
Various lysosmotropic drugs in Figure 1A demonstrated anti-SARS-CoV(-2) efficacy (Dyall
et al., 2014; Zhou et al., 2016; Liu et al., 2020; Weston et al., 2020), offer a more
favorable drug profile than the initially investigated chloroquine and hydroxychloroquine.
Accumulation In Lysosomes of Pulmonary Tissue
Imipramine and chlorpromazine are accumulating in isolated perfused lung tissue and
imipramine in alveolar macrophages (Wilson et al., 1982; Macintyre and Cutler, 1988)
suggesting that lysosomotropic drug concentrations in pulmonary alveoli and protective
effects on SARS-CoV-2 infection of particular drugs are likely. Of the lysosomotropic
in vitro anti-SARS-CoV-2 antibiotics teicoplanin, oritavancin, dalbavancin, and telavancin
(Zhou et al., 2016), solely teicoplanin and telavancin are in accumulating pulmonary
tissue and are expected to be a treatment option.
Additional Therapeutic Benefits In Sars-Cov-2 Infection/Covid-19
Beside lysosomotropism certain intrinsic pharmacological effects are advantageously
in SARS-CoV-2 infection/COVID-19. The incidence of CRS/cytokine storm syndrome associated
with secondary gram-positive bacterial infections is likely to be minimized by using
the pulmonary tissue accumulating antibacterials teicoplanin and telavancin or the
antifungal itraconazole in systemic mycoses in appropriate systemic drug levels.
Choosing A Suitable Route of Administration
Systemic application of chlorpromazine (NCT04366739) and fluoxetine (NCT04377308)
as lysosomotropic drugs may provoke severe and unfavorable adverse effects in mental
healthy patients. Since the respiratory tract is both, the gateway for SARS-CoV-2
infection/COVID-19 and an internal surface, the expedient is a local application in
the airways and/or the respiratory tract. Local application of small molecules is
possible, preferably as inhalant or via nebulizers to avoid (undesirable) systemic
effects. The majority of lysosomotropic drugs should be suitable for inhalation.
Combination With Antivirals and Tmprss2 Inhibitors
COVID-19 originates from a SARS-CoV-2 infection that could not be tackled successfully
by the immune system. The antiviral remdesivir proved to be effective in infection
prophylaxis (phase 0) (de Wit et al., 2020) and viral (SARS-CoV-2) infection (phase
1) within a limited period (5–6 days), shortly after the symptoms emerge and viral
shedding occurs (Mitjà and Clotet, 2020). In severe COVID-19 neither a lower mortality
nor a faster clearance of viruses was observed (Wang et al., 2020). As soon as the
infection initiates a CRS/cytokine storm, it is likely that the transition toward
COVID-19 (phase 2), a disseminated intravascular coagulation/thrombotic microangiopathy,
or a bacterial secondary infection occurs. An effective multi-drug therapy, focusing
on the progression of COVID-19 and emerging severe complications, can be implemented
by lysosomotropic drugs, TMPRSS2 inhibitors and antivirals.
Nafamostat: An In Vivo TMPRSS2 Inhibitor?
Nafamostat is an approved protease inhibitor that inhibits TMPRSS2 (in vitro) (Hoffmann
et al., 2020), prevents (sepsis-related) disseminated intravascular coagulation, and
thrombotic microangiopathy (Okajima et al., 1995; Levi and Thachil, 2020), appears
to be useful in SARS-CoV-2 infection and prophylaxis, and for patients subjected to
extracorporeal circulation such as ECMO (Han et al., 2011). It is doubtful, however,
whether the pulmonary concentration in therapeutically dosage (Ono Pharmaceuticals,
2020) is sufficient to generate a TMPRSS2 inhibition in vivo as demonstrated in vitro
due to poor accumulation in pulmonary tissue (Midgley et al., 1994).
Single or Multi Target Approach: Lysosomotropic Drugs vs. Antibodies
Various clinical trials are currently under way using immunomodulatory IL-1 and IL-6
inhibitors or anti-IL-6R antibodies (anakinra, tocilizumab, siltuximab, and sarilumab)
in patients with COVID-19 (COVID-19 Treatment Guidelines Panel, 2020); limited data,
however, is yet available. In a retrospective study using tocilizumab and hydroxychloroquine,
both demonstrated a limited benefit in survival (Ip et al., 2020). Tocilizumab shortens
mechanical ventilation and hospital stay in severe COVID-19 (Eimer et al., 2020),
while tocilizumab is often accompanied by bacterial pneumonia 2 days after application
(23%) (Pettit et al., 2020).
To improve outcome, antibody cocktails consisting of anti-IL-6, IL-1 receptor blocker,
IL-1 type 1 receptor, and TNF-α are suggested (Harrison, 2020), irrespective of the
risk of serious adverse effects (e.g., bacterial pneumonia) due to more pronounced
interference with the immune defense. Such cocktails are intended to tackle the release
of pro-inflammatory cytokines IL-1β and IL-6 mediating lung and tissue inflammation,
fever, and fibrosis, as they are supposed to be responsible for the emergence of COVID-19.
Although lysosomotropic drugs likewise interfere with the immune defense, such adverse
effects are not reported. In contrast to antibodies, however, only the resynthesis
of IL-6 and thus the available amount is reduced, but not completely obstructed, still
allowing a moderate immune response. Multitargeting on core processes of the viral
infection addressing the formation of multinucleate syncytia and alteration of tissue
structure, ceramide metabolism, and the release of virions could be a key advantage
of lysosomotropic drugs compared to current strategies.
Future Directions
Daunting results of (hydroxy)chloroquine in clinical trials are closely related to
their drug profile and minor lysosomotropism, but not to the mode of action (lysosomotropism)
in general. Observations in patients treated with chlorpromazine and the extensive
accumulation of imipramine in alveolar macrophages and of both, imipramine and chlorpromazine
in isolated perfused lung tissue supports the benefits of lysosomotropic drugs that
are accumulating in pulmonary tissue in SARS-CoV-2 infection/COVID-19.
Promising candidates among lysosomotropic drugs in fact require more than adequate
lysosomotropism; accumulation in pulmonary tissue is a prerequisite as well. It is,
however, likely irrelevant whether the drug or its metabolite(s) is accumulating given
the broad structural requirements for this activity. Since a large number of compounds
has not yet been evaluated for lysosomotropism, many compounds beside those listed
in Figure 1A are expected to meet the requirements described here and may (partially)
be responsible for background immunity to SARS-CoV infection.