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Driving a wedge between viral lipids blocks infection
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There is a massive push to develop new drugs to treat viral infection. Traditional therapeutic strategies aim at viral proteins responsible for each and every step of viral replication. The main drawbacks of these approaches include an ever-increasing pool of drugs specific for a given virus and selection for drug-resistant viruses. An alternative strategy, which has recently gained popularity, targets cellular factors (not limited to viral receptors) involved in virus entry and replication (1, 2). Numerous cellular proteins aiding viral replication have recently emerged from genome-wide screens (3–6), showing the virus’ reliance on various cellular processes. Targeting less variable host factors is an attractive concept that is less prone to selecting for drug-resistant viruses. The flip side of this approach is the potential for serious side effects and the need to target a large and often nonoverlapping number of cellular factors. A study by St. Vincent et al. (7) in PNAS and the paper published earlier by another group (8) introduce an exciting paradigm that focuses on a universal cellular target, which happens to be an intricate part of all enveloped viruses. The authors (7) show that infection by enveloped viruses can be blocked by altering their membrane composition in a way that disfavors their merger with a target cell membrane.
Enveloped viruses surround their nucleocapsids with a host cell-derived lipid membrane and therefore must merge the viral and target cell membranes to initiate new infection. This step is promoted by structurally diverse fusion glycoproteins, which are activated by a specific cellular receptor (or several receptors) and/or acidic endosomal pH (9). Fusion proteins are believed to promote membrane merger by engaging the target membrane, and subsequently, refolding into a stable hairpin structure (Fig. 1A) (10, 11). Because viruses cannot directly use chemical energy released upon ATP or GTP hydrolysis, conformational energy stored in their envelope proteins seems to be the only driving force for membrane fusion (12).
Fig. 1.Viral protein refolding and lipid intermediates en route to membrane fusion. (A) Viral fusion protein refolding into a stable hairpin of trimers, which is coupled to lipid rearrangement through the formation of prehairpin intermediates. (B) Progression of lipid bilayer fusion through the stalk, hemifusion, and fusion pore formation. Lipophilic compounds conferring positive curvature (red triangles) stabilize prefused membranes, preventing the stalk formation and promoting the formation of lytic pores in lipid bilayers. Lipids conferring negative curvature (yellow inverted triangles) augment hemifusion. (C) Negative curvature lipids tend to form an inverted hexagonal HII-phase, whereas positive curvature lipids assemble into micelles.
Depending on the virus, the number of envelope glycoproteins could reach several hundred. The apparent surplus of these proteins reflects, in part, the importance of the fusion step and the nondeterministic nature of this process, which often fails to reach completion. There is evidence that several viral proteins must act in concert to effectively mediate fusion (13–18). Accordingly, the fusion efficiency is known to critically depend on the density of activated viral proteins (13, 19). The above considerations imply that, in general, the energy released from a single viral protein refolding may not be sufficient to destabilize lipid bilayers and promote their fusion. Thus, a synchronous activation and assembly of several fusion proteins into multimeric complexes might help overcome the energy barrier for membrane fusion.
The merger of lipid membranes involves the formation of highly curved (and thus energetically unfavorable) intermediates—stalk, hemifusion, and a fusion pore (Fig. 1B). The main contribution to the overall energy of these intermediates comes from elastic energy of bent monolayers (20), which depends on the intrinsic propensity of lipid sheets to deviate from planarity (described in terms of spontaneous curvature) (21). Lipids with larger polar head groups compared with their hydrocarbon tails confer a positive curvature by bending the membranes away from polar heads (Fig. 1 B and C). By contrast, lipids in which the cross-sectional area of the polar heads is smaller than that of the hydrophobic moiety confer a negative curvature. The merger of contacting monolayers is known to be augmented by negative curvature constituents, whereas lipids favoring the positive membrane curvature disfavor hemifusion (20). The inhibitory effect of positive curvature agents exemplified by lyso-lipids has been shown for diverse fusion reactions mediated by viral and cellular fusion proteins (22). These findings strongly imply that (i) all protein-mediated fusion reactions converge to a common lipid intermediate with a net negative curvature, most likely a hemifusion, and (ii) lipids are essential determinants of the outcome of protein-mediated fusion.
The study by St. Vincent et al. (7) introduces a class of wedge-shaped rigid amphipathic fusion inhibitors (RAFIs) that block infectivity of unrelated enveloped viruses, apparently through conferring a positive curvature to their lipid membranes. Like lyso-lipids, RAFIs seem to counteract the well-balanced action of fusion proteins through the lipid phase, without directly interacting with viral proteins. An important feature of RAFIs, as well as other compounds that target the viral membrane (8), is that these are not toxic for cells. The selective effect of RAFIs on the metabolically inactive viral membrane most likely originates from the lack of membrane repair mechanisms, which are effective in cells. As expected for compounds that alter the propensity of viral lipids to undergo fusion, RAFIs blocked infection by several enveloped viruses but did not affect the infectivity of nonenveloped viruses at much higher doses.
The overall shape of RAFIs and their ability to disfavor the transition from a lamellar to an inverted hexagonal phase (highly curved inverted lipid cylinders) (Fig. 1C) are consistent with the notion that these molecules confer a positive curvature to viral lipids, thereby antagonizing the action of viral fusion proteins. These results provide an exciting proof-of-concept for developing broad-spectrum entry inhibitors that could block fusion of virtually all enveloped viruses. Importantly, this class of drugs is unlikely to select for resistant variants, because viruses have virtually no control over their lipid composition.
Although the study by St. Vincent et al. (7) is an important milestone for future antiviral strategies, a number of questions
RAFIs blocked infection by several enveloped viruses but did not affect the infectivity of nonenveloped viruses.
remain unanswered. It is not completely clear yet whether altering the membrane curvature is the only or even the main mechanism of the RAFIs’ effect on enveloped viruses. Certain features of these compounds reported in this study might be indicative of additional modes of action. First, whereas amphipathic molecules conferring positive curvature lyse membranes by favoring the formation of lipidic pores (Fig. 1B), high concentrations of RAFIs did not seem to damage cells or lyse viruses. It is worth pointing out, however, that the results presented by St. Vincent et al. (7) argue against dissolution of the viral membrane by RAFIs but do not rule out the membrane permeabilizing effect. Second, the predominantly polar nature of amphipaths conferring positive curvature is manifested in a relatively high critical micelle concentration and the ease of their extraction from membranes on washing (13). In contrast, RAFIs seem to incorporate into viral membranes virtually irreversibly, as evidenced by their long-lasting inhibitory effect on pretreated viruses.
Further studies are needed to fully elucidate the mechanism of inhibition of viral fusion by RAFIs and related compounds. For instance, it would be interesting to determine the molar fraction of RAFIs in the viral membrane at an inhibitory concentration. This could help evaluate the corresponding change in spontaneous curvature based on the shift in the temperature of lamellar to inverted hexagonal phase transition measured in this study. Irrespective of the exact mechanism of action, the low cytotoxic effect of this class of viral fusion inhibitors might prove decisive for their future clinical applications.
There is a massive push to develop new drugs to treat viral infection. Traditional therapeutic strategies aim at viral proteins responsible for each and every step of viral replication. The main drawbacks of these approaches include an ever-increasing pool of drugs specific for a given virus and selection for drug-resistant viruses. An alternative strategy, which has recently gained popularity, targets cellular factors (not limited to viral receptors) involved in virus entry and replication (1, 2). Numerous cellular proteins aiding viral replication have recently emerged from genome-wide screens (3–6), showing the virus’ reliance on various cellular processes. Targeting less variable host factors is an attractive concept that is less prone to selecting for drug-resistant viruses. The flip side of this approach is the potential for serious side effects and the need to target a large and often nonoverlapping number of cellular factors. A study by St. Vincent et al. (7) in PNAS and the paper published earlier by another group (8) introduce an exciting paradigm that focuses on a universal cellular target, which happens to be an intricate part of all enveloped viruses. The authors (7) show that infection by enveloped viruses can be blocked by altering their membrane composition in a way that disfavors their merger with a target cell membrane.
Enveloped viruses surround their nucleocapsids with a host cell-derived lipid membrane and therefore must merge the viral and target cell membranes to initiate new infection. This step is promoted by structurally diverse fusion glycoproteins, which are activated by a specific cellular receptor (or several receptors) and/or acidic endosomal pH (9). Fusion proteins are believed to promote membrane merger by engaging the target membrane, and subsequently, refolding into a stable hairpin structure (Fig. 1A) (10, 11). Because viruses cannot directly use chemical energy released upon ATP or GTP hydrolysis, conformational energy stored in their envelope proteins seems to be the only driving force for membrane fusion (12).
Viral protein refolding and lipid intermediates en route to membrane fusion. (A) Viral fusion protein refolding into a stable hairpin of trimers, which is coupled to lipid rearrangement through the formation of prehairpin intermediates. (B) Progression of lipid bilayer fusion through the stalk, hemifusion, and fusion pore formation. Lipophilic compounds conferring positive curvature (red triangles) stabilize prefused membranes, preventing the stalk formation and promoting the formation of lytic pores in lipid bilayers. Lipids conferring negative curvature (yellow inverted triangles) augment hemifusion. (C) Negative curvature lipids tend to form an inverted hexagonal HII-phase, whereas positive curvature lipids assemble into micelles.
Depending on the virus, the number of envelope glycoproteins could reach several hundred. The apparent surplus of these proteins reflects, in part, the importance of the fusion step and the nondeterministic nature of this process, which often fails to reach completion. There is evidence that several viral proteins must act in concert to effectively mediate fusion (13–18). Accordingly, the fusion efficiency is known to critically depend on the density of activated viral proteins (13, 19). The above considerations imply that, in general, the energy released from a single viral protein refolding may not be sufficient to destabilize lipid bilayers and promote their fusion. Thus, a synchronous activation and assembly of several fusion proteins into multimeric complexes might help overcome the energy barrier for membrane fusion.
The merger of lipid membranes involves the formation of highly curved (and thus energetically unfavorable) intermediates—stalk, hemifusion, and a fusion pore (Fig. 1B). The main contribution to the overall energy of these intermediates comes from elastic energy of bent monolayers (20), which depends on the intrinsic propensity of lipid sheets to deviate from planarity (described in terms of spontaneous curvature) (21). Lipids with larger polar head groups compared with their hydrocarbon tails confer a positive curvature by bending the membranes away from polar heads (Fig. 1 B and C). By contrast, lipids in which the cross-sectional area of the polar heads is smaller than that of the hydrophobic moiety confer a negative curvature. The merger of contacting monolayers is known to be augmented by negative curvature constituents, whereas lipids favoring the positive membrane curvature disfavor hemifusion (20). The inhibitory effect of positive curvature agents exemplified by lyso-lipids has been shown for diverse fusion reactions mediated by viral and cellular fusion proteins (22). These findings strongly imply that (i) all protein-mediated fusion reactions converge to a common lipid intermediate with a net negative curvature, most likely a hemifusion, and (ii) lipids are essential determinants of the outcome of protein-mediated fusion.
The study by St. Vincent et al. (7) introduces a class of wedge-shaped rigid amphipathic fusion inhibitors (RAFIs) that block infectivity of unrelated enveloped viruses, apparently through conferring a positive curvature to their lipid membranes. Like lyso-lipids, RAFIs seem to counteract the well-balanced action of fusion proteins through the lipid phase, without directly interacting with viral proteins. An important feature of RAFIs, as well as other compounds that target the viral membrane (8), is that these are not toxic for cells. The selective effect of RAFIs on the metabolically inactive viral membrane most likely originates from the lack of membrane repair mechanisms, which are effective in cells. As expected for compounds that alter the propensity of viral lipids to undergo fusion, RAFIs blocked infection by several enveloped viruses but did not affect the infectivity of nonenveloped viruses at much higher doses.
The overall shape of RAFIs and their ability to disfavor the transition from a lamellar to an inverted hexagonal phase (highly curved inverted lipid cylinders) (Fig. 1C) are consistent with the notion that these molecules confer a positive curvature to viral lipids, thereby antagonizing the action of viral fusion proteins. These results provide an exciting proof-of-concept for developing broad-spectrum entry inhibitors that could block fusion of virtually all enveloped viruses. Importantly, this class of drugs is unlikely to select for resistant variants, because viruses have virtually no control over their lipid composition.
Although the study by St. Vincent et al. (7) is an important milestone for future antiviral strategies, a number of questions
RAFIs blocked infection by several enveloped viruses but did not affect the infectivity of nonenveloped viruses.
remain unanswered. It is not completely clear yet whether altering the membrane curvature is the only or even the main mechanism of the RAFIs’ effect on enveloped viruses. Certain features of these compounds reported in this study might be indicative of additional modes of action. First, whereas amphipathic molecules conferring positive curvature lyse membranes by favoring the formation of lipidic pores (Fig. 1B), high concentrations of RAFIs did not seem to damage cells or lyse viruses. It is worth pointing out, however, that the results presented by St. Vincent et al. (7) argue against dissolution of the viral membrane by RAFIs but do not rule out the membrane permeabilizing effect. Second, the predominantly polar nature of amphipaths conferring positive curvature is manifested in a relatively high critical micelle concentration and the ease of their extraction from membranes on washing (13). In contrast, RAFIs seem to incorporate into viral membranes virtually irreversibly, as evidenced by their long-lasting inhibitory effect on pretreated viruses.
Further studies are needed to fully elucidate the mechanism of inhibition of viral fusion by RAFIs and related compounds. For instance, it would be interesting to determine the molar fraction of RAFIs in the viral membrane at an inhibitory concentration. This could help evaluate the corresponding change in spontaneous curvature based on the shift in the temperature of lamellar to inverted hexagonal phase transition measured in this study. Irrespective of the exact mechanism of action, the low cytotoxic effect of this class of viral fusion inhibitors might prove decisive for their future clinical applications.
Footnotes
To understand how sodium bicarbonate works, it is helpful to first understand the concept of pH.
How pH Affects Exercise Performance
In chemistry, pH is a scale used to grade how acidic or alkaline (basic) a solution is.
A pH of 7.0 is considered neutral. Anything lower than 7.0 is acidic and anything above that is alkaline.
As humans, our pH is naturally close to neutral. It normally stays around 7.4 in blood and 7.0 in muscle cells.
You function best when your acid-alkaline balance remains close to this target, which is why your body has various ways to maintain these levels.
However, certain diseases or external factors can disrupt this balance. One of these factors is high-intensity exercise, also known as anaerobic exercise (
During anaerobic exercise, your body’s demand for oxygen exceeds the available supply. As a result, your muscles cannot rely on oxygen to produce energy.
Instead, they must switch to a different pathway — the anaerobic pathway.
Creating energy through the anaerobic pathway produces lactic acid. Too much lactic acid decreases your muscle cells’ pH below the optimal 7.0 (
This disrupted balance limits energy production and may also reduce your muscles’ ability to contract. Both of these effects ultimately lead to fatigue, which reduces exercise performance (
How Sodium Bicarbonate Helps Maintain pH
Sodium bicarbonate has an alkaline pH of 8.4 and can therefore raise your blood pH slightly.
Higher blood pH allows acid to move from muscle cells into the bloodstream, returning their pH to 7.0. This enables the muscles to continue contracting and producing energy (
Scientists believe this is the primary way that sodium bicarbonate can help you exercise harder, faster or for longer (
BOTTOM LINE:Sodium bicarbonate clears acid out of muscle cells, helping restore an optimal pH. This may decrease fatigue and increase performance.
Scientists have examined how sodium bicarbonate affects exercise performance for more than 8 decades.
Abstract
Emerging links between endosomal pH and cancer
https://www.researchgate.net/publication/340473866_Emerging_links_between_endosomal_pH_and_cancer
Extracellular acidification is a well-known driver of tumorigenesis that has been extensively studied. In contrast, the role of endosomal pH is novel and relatively unexplored. There is emerging evidence from a growing number of studies showing that the pH of endosomal compartments controls proliferation, migration, stemness, and sensitivity to chemoradiation therapy in a variety of tumors. Endosomes are a crucial hub, mediating cellular communication with the external environment. By finely regulating the sorting and trafficking of vesicular cargo for degradation or recycling, endosomal pH determines the fate of plasma membrane proteins, lipids, and extracellular signals including growth factor receptors and their ligands. Several critical regulators of endosomal pH have been identified, including multiple isoforms of the family of electroneutral Na+/H+ exchangers (NHE) such as NHE6 and NHE9. Recent studies have shed light on molecular mechanisms linking endosomal pH to cancer malignancy. Manipulating endosomal pH by epigenetic reprogramming, small molecules, or nanoparticles may offer promising new options in cancer therapy. In this review, we summarize evidence linking endosomal pH to cancer, with a focus on the role of endosomal Na+/H+ exchangers and how they affect the prognosis of cancer patients, and also suggest how regulation of endosomal pH may be exploited to develop new cancer therapies.
Reversible conformational change in herpes simplex virus glycoprotein B with fusion-from-without activity is triggered by mildly acidic pH
Virology Journal 7, Article number: 352 (2010)
Abstract
https://link.springer.com/article/10.1186/1743-422X-7-352
Lysosomotropic agents, which elevate intravesicular pH, block HSV entry by trapping virions in endocytic compartments [4, 6]. Pretreatment of isolated HSV particles with mildly acidic pH inactivates viral entry activity, which is a characteristic of viruses that are directly triggered by endosomal pH for fusion [4]. Low pH together with soluble gD-receptor triggers association of HSV with artificial membranes [7].
If Hydroxychloroquine and Chloroquine are able to change the pH of your lysosomes, I don't see why it wouldn't be possible to do this with other methods (like baking soda and lime juice).
https://www.sciencedirect.com/science/article/pii/S0898656820301832
Fig. 1. CQ and HCQ de-acidify acidic organelles.
Membrane bound vesicles in the endocytic pathway (early endosome, recycling endosome, late endosome, and lysosomes) and the biosynthetic secretory pathway (Golgi apparatus and secretory vesicles) all display varying degrees of acidity, and these vesicles rapidly acidify as they progress along the endocytic or secretory pathway. As diprotic weak bases, CQ and HCQ are taken up by cells and trapped in these acidic organelles, where they neutralize pH and alter their structure, function, and trafficking.
CQ and its analogues HCQ (Fig. 1) are concentrated in acidic endolysosomes [[66], [67], [68]] where they neutralize endolysosome pH [20,55], induce markedly enlargement of endolysosomes [69,70], change the positioning of endolysosomes from perinuclear to the periphery of cells [48,71], and lead to lysosome membrane permeabilization [54,72,73]. CQ-induced endolysosome de-acidification results in the accumulation and aggregation of undegraded substrates and atypical cleavages that lead to generation of toxic intermediates [42,74]. CQ-induced lysosomal membrane permeabilization leads to the translocation of lysosomal contents (eg. cathepsins) to the cytoplasm and to the induction of mitochondria damage and cell death [75,76]. CQ-induced endolysosome de-acidification also impairs vesicular fusion and inhibits autophagic flux by decreasing autophagosome-lysosome fusion [70,77]. In addition, CQ enhances lysosome exocytosis [72,78] and the release of exosomes [79]. However, CQ does not affect endocytosis [70].
Golgi apparatus helps process, sort and traffic proteins and lipids destined for secretion, membranes, and organelles. Sub-compartments of Golgi are mildly acidic; pH values range from 6.7 at cis-Golgi to 6.0 at trans-Golgi [29]. Secretory vesicles (constitutive or regulated) are more acidic; luminal pH ranges from 5.2 to 5.7 [23,24]. Consistent with the view that an acidic environment is critical for the processing of proteins and lipids, deacidification of Golgi results in defects in posttranslational modifications and processing of secreted proteins. For example, glycosylation is pH-sensitive [80] and an increase of 0.2 pH units results in decreased glycosylation [81]. In terms of sorting and trafficking of proteins and lipids, deacidification impaired anterograde transport from Golgi to secretary vesicles [82], retrograde transport from Golgi back to the endoplasmic reticulum (ER) [83], the delivery of lysosomal hydrolases to lysosomes via mannose-6-phosphate receptor (M6PR) [78,84], the integrity of Golgi itself [85,86], and the sorting and proteolytic maturation of prohormones in secretory granules [87]. Thus, it is not surprising that defective Golgi pH regulation has been implicated in a number of human diseases including autosomal recessive Cutis Laxa type II [88] and multigenerational non-syndromic intellectual disability [89].
Similar to endolysosomes, CQ and HCQ (Fig. 1) are concentrated in and neutralize the pH of acidic Golgi [29], and induce marked dilatation of the Golgi cisternae [90]. Functionally, CQ-induced de-acidification results in glycosylation deficits [81], deficits in the formation of functional transport vesicles, and the inability of budding vesicles to pinch off and form functional transport vesicles [91,92]. CQ also changed distribution patterns of mannose-6-phosphate receptors and decreased the delivery of lysosomal enzymes into lysosomes via mannose-6-phosphate receptors [93]; the latter process might be responsible for CQ-induced changes in lysosome exocytosis [72,78]. Furthermore, CQ-induced de-acidification also leads to deficits in sorting and proteolytic maturation of the prohormones pro-somatostatin [94], adrenocorticotropic hormone (ACTH) [95], and pro-insulin [96].
3. Coronaviruses and organellar pH
Coronaviruses are single-stranded RNA virus that are enveloped with crown-like spikes on the surface. Various types of human coronaviruses cause acute lung injury and acute respiratory distress syndrome that results in pulmonary failure; these include severe acute respiratory syndrome coronavirus (SARS-CoV), H5N1 influenza A (H1N1), Middle East respiratory syndrome coronavirus (MERS-CoV), and most recently SARS-CoV-2 the root cause of the current COVID-19 pandemic [109]. Unlike the malaria-causing parasite that has its own acidic digestive vacuole, viruses use host cell mechanisms for entry and replication. Many viruses are endocytosed into endolysosomes following interactions with cell surface proteins, lipids and sugar moieties [110].
Like other enveloped viruses, SARS-CoV-2 enters host cells and utilizes host cell machinery for replication. The spiked glycoprotein on the outer surfaces of coronaviruses are responsible for the attachment and entry of the virus to host cells via receptor-mediated endocytosis with the assistance of angiotensin-converting enzyme 2 (ACE2) [7,8,111] and possibly other co-receptors [112]. Once inside endosomes, SARS-CoV-2 is either metabolized by pH-sensitive hydrolytic enzymes or it escapes from these organelles. For SARS-CoV [[113], [114], [115], [116], [117], [118], [119]], pH-dependent, furin or cathepsin L-mediated cleavage of spike envelope protein appears to facilitate viral envelope fusion with endosomes and the virus with its genomic contents are released into the cytoplasm of the host cell. Similar to SARS-CoV [120], SARS-CoV-2 replication occurs in the cytoplasm and it may assemble and mature in Golgi from which it is released via secretary vesicles (Fig. 2).
Fig. 2. Coronaviruses and organellar pH.
SARS-CoV enters host cells via endocytosis and utilizes host cell machinery for replication. Once inside endosomes, SARS-CoV escapes from these organelles via pH- and cathepsin L-dependent mechanism. Following replication in the cytoplasm SARS-CoV may assemble and mature in trans-Golgi, from which it is released via secretary vesicles. By de-acidifying these acidic organelles, CQ and HCQ may block virus entry and affect post-translational modifications including the proteolysis and glycosylation of SARS-CoV.
Other enveloped viruses like influenza A and Ebola, also use the acidic environment of endosomes or endolysosome hydrolases to drive the fusion of viral membranes with endosome membranes and the release of viral genomic content into the cytoplasm [121]. As such, endolysosome de-acidification with a v-ATPase inhibitor [122] or CQ [123,124] has been used frequently to inhibit cellular entry of enveloped virus in vitro. Once replicated in the cytoplasm, some viruses are packaged in trans-Golgi network where low pH facilitates the maturation of the virus. CQ, by de-acidifying Golgi, impairs the maturation of viruses and decreases viral infection, in part, by increasing the accumulation of, for example, non-infectious herpes simplex virus 1 particles [125], HIV-1 [126] and flavivirus [[126], [127], [128], [129]]. Currently it is not clear whether CQ affects membrane invagination and viral packing into the trans-Golgi network or the extracellular release of mature virus.
Given the above findings, it is not surprising that CQ and HCQ are being tested for their possible effectiveness against SARS-CoV-2. Preliminary in vitro studies have shown that both CQ [9] and HCQ [10,11] exhibit antiviral effects against SARS-CoV-2. Although the underlying mechanisms are not fully understood, it is possible that endolysosome de-acidification by CQ (Fig. 2) may block pH-dependent, furin - or cathepsin L-mediated cleavage of the spike envelope protein that facilitates viral envelope fusion with endosome membranes [115,116,118,119]. Further, CQ-induced Golgi de-acidification may result in decreased expression levels of ACE2. Such mechanisms have been implicated previously with SARS-CoV; CQ decreased the binding of SARS-CoV spike protein with ACE2 [8] and CQ-induced Golgi de-acidification (Fig. 2) affected post-translational modifications including the proteolysis and glycosylation of SARS-CoV virions [8].
https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202000654R
The lysosome: A potential juncture between SARS‐CoV‐2 infectivity and Niemann‐Pick disease type C, with therapeutic implications
3 NPC‐INDUCED ABERRATIONS IN CATHEPSIN LOCALIZATION AND ACTIVITY INTERFERE WITH VIRAL FUSION
In addition to their defective egress of intra‐lysosomal cholesterol, NPC cells are also known to have impaired localization and activities of various lysosomal enzymes, including cathepsins L and B, a finding also reported in other LSDs.41, 42 Specifically, the chronic accumulation of various substances within the lysosomes of NPC cells, with sphingomyelin and sphingosine being notable examples of these substances, has been found to disrupt the integrity of the lysosomal membrane, leading to the leakage of several lysosomal enzymes, such as cathepsins, into the cytosol.41, 43-45 Other studies have also shown direct inhibitory effects of the lipids accumulating in NPC toward lysosomal cathepsins.46 Moreover, the same lipid substrates that accumulate within the lysosomes of LSD cells, have also been shown to disrupt normal acidification of the lysosomes, thereby increasing the intra‐lysosomal pH and adversely affecting the activities of enzymes within those lysosomes.47-50 In fact, these substrates have also been found to simultaneously impede normal fusion of vesicles transporting lysosomal cargo, including enzymes, such as cathepsins, into the lysosome. This further depletes the intra‐lysosomal stores of key hydrolytic enzymes.41, 47, 51 Thus, it is also reasonable to speculate that these NPC‐related aberrations in lysosomal enzyme localization, transport into the lysosomes, and intracellular activity levels, especially those of cathepsins B and L, pose additional “barriers” in NPC that could prevent the trafficking and intracellular processing of viral membrane proteins, a step that is required for successful viral fusion. Therefore, it may also be worth testing various inhibitors of cathepsins B and L, such as recombinant cystatins or stefins, for their therapeutic potential against SARS‐CoV‐2 infection, given their ability to induce an NPC‐resembling lysosomal dysfunctional state.17, 18, 52
5.1 Chloroquine ± Azithromycin
Among the various repurposed drugs currently under investigation for the treatment of COVID‐19, chloroquine and its derivatives, such as hydroxychloroquine, emerged as the first potentially efficacious existing drug for COVID‐19, based on documented pre‐clinical efficacy and expert consensus by several Chinese scientific authorities.61-63 As a result, there has been cumulative interest in testing the efficacy of chloroquine and its derivatives, in the treatment of COVID‐19, such that there are now, over 20 different related clinical trials in trial registries.64 Additionally, two different phase III clinical trials are currently investigating the use of hydroxychloroquine for pre‐ and post‐exposure prophylaxis against COVID‐19 in healthcare workers (NCT04303507 and NCT04328285). However, it remains unclear how an anti‐malarial drug‐like chloroquine could also exert antimicrobial activity against a viral pathogen‐like SARS‐CoV‐2, raising the question of whether the same mechanism underlying chloroquine's antimalarial activity may also be responsible for its antiviral effects.
Importantly, chloroquine has been used for many years in lysosomal storage disease (LSD) research, given its ability to inhibit lysosomal fusion with endosomes, as well as inhibit the activity of various lysosomal enzymes. These properties of chloroquine allowed it to be used in vitro to pharmacologically induce transient LSD‐like cellular pathology.65-67 In fact, this lysosomotropic activity of chloroquine is what is thought to be responsible for its anti‐malarial mode of action. Specifically, chloroquine is believed to undergo trafficking into the lysosomes of Plasmodium trophozoites, where it gets protonated and entrapped, thereby disrupting the fusion of these lysosomes with the “food vacuoles” (ie, phagosomes) of the trophozoites, hampering the latter's ability to feed on engulfed red blood cells.68, 69 However, this same propensity of chloroquine to traffic into, and concentrate within intracellular acidic organelles, also cross‐reacts with mammalian cells, inducing similar disruptions in the functions of their lysosomes as the ones it induces for protozoal food vacuoles, that is, interfering with endo‐lysosomal fusion, elevating intra‐lysosomal pH, and inducing partial permeabilization of lysosomal membranes, which altogether mirror the lysosomal pathology intrinsic to several LSDs.65, 69, 70
Such lysosomal “disruptions” are actually intrinsic to NPC in particular, as previously discussed, which further supports the possibility of a lysosome‐mediated antiviral activity for chloroquine against SARS‐CoV‐2, since chloroquine is capable of inducing transient NPC‐like lysosomal abnormalities that may interfere with intracellular viral trafficking and fusion.
In support of this hypothesis, previous studies have successfully shown that the antiviral activity of chloroquine against several caliciviridae, another family of RNA viruses, occurs through chloroquine's ability to inhibit cathepsin L.71 Furthermore, chloroquine also inhibits the transport of cholesterol out of the lysosomes, including to the plasma membrane, which would be expected to reduce the abundance, and alter the composition of membrane rafts,72 thereby mimicking the raft alterations seen in NPC. Additionally, chloroquine has also been shown to interfere with the trimming of the N‐glycosylated side chain of ACE2, which may affect the internalization of ACE2, and subsequently, viral entry.73 Interestingly, N‐glycosylation has actually been shown to be altered in NPC,74 further suggesting that NPC cells likely possess inherent chloroquine‐like effects of altered N‐glycosylation modification of ACE2, which potentially further offers these cells with “protection” against SARS‐CoV‐2 infection. In that regard, a small open‐label non‐randomized clinical trial conducted in France (EU CTR 2020‐000890‐25) has recently gained considerable interest, after it showed a statistically significant difference in the rates of SARS‐CoV‐2 viral clearance from the nasal swabs of COVID‐19‐positive patients receiving a combination of hydroxychloroquine and azithromycin, the latter being a macrolide antibiotic, compared with those receiving hydroxychloroquine only (P = .002 at Day 3 post‐inclusion), or no antimicrobial therapy whatsoever (P = .005 at Day 3 post‐inclusion).75 In this context, it is also important to highlight the lysosomotropic activity of azithromycin itself, the drug combined with hydroxychloroquine in that trial, as an add‐on therapy.75 Similar to chloroquine or its derivatives, azithromycin also undergoes trafficking to, and accumulation within the lysosomes, where it alkalinizes the luminal pH of these organelles, thereby inhibiting the activity of resident enzymes.76 In fact, the combination of both drugs, chloroquine and azithromycin, has been previously shown to exhibit synergistic lysosomotropic effects, especially with regards to increasing lysosomal pH.76 Moreover, chronic azithromycin treatment in patients with cystic fibrosis has been shown to increase susceptibility to mycobacterial infections, which usually rely heavily on adequate phagocytosis and bacterial containment within phagosomes.77 Azithromycin has been particularly shown to block the lysosome‐mediated acidification of phagosomes containing the mycobacteria, allowing the latter to escape the phagosomes and multiply uncontrollably.77 However, in contrast to mycobacteria where the intact lysosomal function is required to contain/control the infection,78, 79 in SARS‐CoV‐2 infections, the intact lysosomal function is actually needed for successful viral fusion and establishment of infection, as discussed earlier. Thus, it is possible that the observed synergistic efficacy of azithromycin combination with hydroxychloroquine, in the treatment of SARS‐CoV‐2, is the result of their similar lysosome‐mediated antiviral activities, that is, their independent inhibition of endosomal‐lysosomal fusion and lysosomal proteases, which are key for successful viral fusion. In addition, azithromycin's tropism toward the lysosomes has also been shown to induce an accumulation of neutral lipids, namely free cholesterol and phospholipids, within these organelles,80, 81 which phenocopies the “natural” cellular phenotype of NPC cells.23
5.5 Cepharanthine
Finally, another repurposed drug being tested against COVID‐19 is cepharanthine, a plant‐derived alkaloid with prominent anti‐inflammatory effects.118 In fact, cepharanthine demonstrated the highest potency among 2406 different clinically approved drugs that were screened against COVID‐19, with preclinical data suggesting it targets the entry of SARS‐CoV‐2.118 Interestingly, cepharanthine has also been shown to undergo intracellular trafficking to the lysosomes, where it physically interacts with and inhibits the NPC1 protein, resulting in lysosomal cholesterol accumulation and elevated intra‐lysosomal pH.119 It is, therefore, possible that cepharanthine's exhibited activity against SARS‐CoV‐2 is mediated, at least partially, by its lysosomotropic effects of directly inhibiting the NPC1 protein and inducing a cellular phenocopy of NPC.
6 CONCLUSION
To summarize, this report raises the hypothesis that the intracellular biochemical abnormalities inherent to LSDs in general and NPC in particular, may pose an “unfavorable” host cell environment for the entry, trafficking, and fusion of SARS‐CoV‐2. Specifically, we postulate that the altered composition of the plasma membrane and lipid rafts in NPC may affect the trafficking of ACE2, the primary host cell membrane receptor responsible for viral docking, thereby interfering with viral infection. Moreover, the increased levels of ADAM17 in the plasma membrane of NPC cells promote ACE2 shedding, thereby inhibiting viral docking at the plasma membrane of host cells. Additionally, the NPC‐related lysosomal membrane permeabilization, which leads to cathepsin L leakage, and the increased intra‐lysosomal pH seen in NPC, impair the activity of cathepsin L, a key protease required for the successful fusion of SARS‐CoV‐2. Furthermore, we highlight how two key oxysterols whose levels are notably elevated in NPC, 25‐HC, and 7‐KC, possess potent antiviral activities, which further grants NPC cells the characteristic of being an unfavorable host cell environment for successful SARS‐CoV‐2 infectivity. We also discuss how the different repurposed drugs demonstrating preliminary efficacy in the treatment of COVID‐19 (chloroquine, azithromycin, remdesivir, triazoles, glycopeptide antibiotics, and cepharanthine) possess lysosomotropic activities, which we propose as being the unifying mechanism underlying their demonstrated and shared antiviral activity against SARS‐CoV‐2. Overall, we propose that pharmacologically targeting one or more of the metabolic facets that comprise the NPC cellular phenotype, may prove beneficial in identifying and rapidly developing treatments for COVID‐19 (Figure 1).
https://www.avensonline.org/wp-content/uploads/JOBY-2377-987X-07-0058.pdf
pH, Presents a New Weapon in the Battle Against COVID-19
Introduction As of March 2020, according to the report from recent news, an estimated more than 1.7 billion people around the world had been ordered to “Stay-at-Home” or affected by country lockdown because of COVID-19 [1]. People are living in the lockdown scenario continuously exposed to the unknown condition of the virus. We need to supply Personal Protective Equipment (PPE) and disinfectant materials such as detergents, alcohol and surfactants to individuals as quickly as possible. However, with the current rapid spreading rate of COVID-19 globally, PPEs, disinfectants, and related protocols are challenging to arrive in time. Besides the cleanse of the coronavirus on rough surfaces and human skin, sometimes we need to remove the potential coronavirus resided on the delicate surface of items such as fresh-produces and foods. For the 1.7 billion population in lockdown, it is vital to provide fresh and active food without any concern while keeping the waste materials at minimum is essential at this critical time. Lack of essential nutrients, vitamins, and trace elements make the immune system weaken and suspect more human beings against COVID-19 [2]. Information from the structure of COVID-19 pointed out that this specific virus, SARS-CoV-2, contains a lipid envelope, which keeps this coronavirus invincible [3]. The entry mechanism of SARSCoV-2 in COVID-19 uses S-1 spike proteins to attach to numerous ACE receptors in the human respiratory tract. ACE-2 receptors on the human lungs are the primary docking site for COVID-19’s S-1 spike protein to attach [3]. In recent study on the virus structure of COVID-19, which SARS-CoV-2 belongs to the beta coronavirus subtype [4]. The virus has a round outer lipid bilayer membrane that has a diameter of approximately 60-140 nm [4]. Like other coronaviruses, SARS-CoV-2’s lipid bilayer membrane is sensitive to UV and heat treatments. Furthermore, the SARS-CoV-2 virus in COVID-19 can be inactivated effectively by lipid solvents such as ether (75%) solution, ethanol, chlorine disinfectant, peroxyacetic acid, and some chloroform [4]. Applying the method to sterilization, such as the use of detergent, surfactant, UV, and heat can deteriorate precious produces, food, and other delicate materials. Instead, we need to find alternative methods to remove SARS-CoV-2 from the delicate surfaces. Listed studies and reviews have pointed out how an alteration in pH level can affect virus activities in human body (Table 1). The extreme acidic or basic environment can potentially inhibit the virus’s function on stability and transmission. In the study human coronavirus 229E was diluted 10-fold in buffers at different pH levels and incubated for 6 hours [5].
They found out that the optimal stability of the virus was at pH 6, at both 4 °C and 33 °C. However, when the virus is at extreme pH levels, it was more stable when incubated at 4 °C. When they exposed the virus in pH 4 or pH 9 at 33 °C, viral infectivity was not detectable. Another investigation discovered that this coronavirus was inactivated by the use Ultraviolet Light (UV), use of heat treatment 65 °C or higher, place the virus in alkaline (pH>12) or acidic (pH<3) conditions, also treat the virus with formalin and glutaraldehyde [6]. A study indicated that the survival rate of coronavirus SARS COV-1 to host cells was affected by low pH levels and warm temperatures [7,8]. The activity of coronavirus decreases when the pH is lower than neutral (pH<7.0) and when the temperature increase from 20 °C to 37 °C [7]. This study can be useful when dealing with COVID-19 since the stability and functionalities are similar to SARS-CoV-1 (Figure 1). In our daily life, a wide range of acidic fluids with different pH levels ranging from pH 2.0 ~ pH 5.0 was available to buy and use. Orange or grapefruit juice (pH=3.0), acetic acid/lemon juice (pH=2.0), citric acid/vinegar (pH=2.0), and Coke and Pepsi (pH=2.5) [9]. These acidic fluids are cheap, affordable, easier to obtain by normal civilians. According to a study on HIV prevention and treatment by using lemon juice and lime juice, Short et al. pointed out that when acidic solution mixed with human serums, the combined pH level
around 4.0~4.3 can prevent HIV infecting the human host [8]. They also mentioned a study carried out by the team in 2004 on the use of fresh lemon or lime juice (pH=2.3 and 2.4 respectively) at 20% concentration could inactivate viruses [10]. The protocol uses filtered pools of lemon juice (pH 2.3) and lime juice (pH 2.4), which added to a selection of various culture medium of HIV-BaL viruses. They confirmed that 20% concentration of lime or lemon juice reduced the pH to 2.9, 90% of the viruses are inactivated in 2 min. When they observe the culture medium in a 10% concentration of lemon or lime juice (pH 3.4–3.7), only 50% of the viruses are inactivated in 2 min [10]. In further discussion, mentioned that a clinical trial on lemon juice or lime juice used for HIV treatment should be conducted ethically with an effective protocol to lower HIV spreading [8]. Furthermore, we can utilize these acidic fluids to test the activity of the novel Coronavirus (COVID-19) at a set range of temperatures (20 °C~37 °C) and in a duration time exposure to observe the outcome. This proposed experiment may eventually help to solve a low quantity of PPEs and disinfectants available in certain countries
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660974/
Sodium bicarbonate nanoparticles modulate the tumor pH and enhance the cellular uptake of doxorubicin
While the main site of bicarbonate activity seems to be in the extracellular matrix, intracellularly, the bicarbonate liposomes may neutralize the endosomal pH, triggering cytosolic release of doxorubicin as well, similarly to the ‘proton sponge’ theory [87]. Having this said, we believe the dominant mechanism is drug release outside the cell.
Tumor pH
The intra-tumoral pH was measured 24 hr after an intravenous injection of liposomal bicarbonate to BALB/c mice bearing orthotopic triple negative breast cancer tumors. We chose to measure the pH at this time point since the liposomal accumulation has been shown to peak then in the tumor post IV injection [25, 27]. The pH value in the liposomal bicarbonate-treated group was 7.38±0.04 compared to 7.13±0.06 in the untreated tumor (Figure 5). pH measurement of healthy mammary fat pad had a physiological value of 7.46±0.01. These data indicate that liposomal bicarbonate can elevate the tumor pH. Each tumor was measured in three different sites (as mentioned in the Methods section): two peripheral points and one measurement in the tumor core. For the untreated group, the average of all the measurements was 7.13±0.06, while the pH value measured in the tumor core was 6.89±0.03, compared to 7.3±0.04 in the peripheral measurements. These results demonstrate the ability to affect tumor pH using liposomal bicarbonate. While the differences in pH values between the treated and untreated groups may seem minor, the corresponding proton concentration alterations are much more significant and can affect the protonation state of doxorubicin molecules. Using the Hendrson–Hasselblach equation (pKa of doxorubicin is 8.2) [88], the unionized form of doxorubicin is 76% greater at pH 7.38 compared to 7.13, which is reflected by increased cellular uptake of the drug.
pH sensing via bicarbonate-regulated “soluble” adenylyl cyclase (sAC)
Nawreen Rahman
- Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA
https://www.frontiersin.org/articles/10.3389/fphys.2013.00343/full
pH Sensing in the Endosomal-Lysosomal Pathway
The endo-lysosomal system is central to the processes of autophagy and endocytosis (Klionsky, 2007; Mizushima, 2007), and there is growing appreciation of its involvement in a broad range of diseases (Futerman and Van Meer, 2004; Nixon et al., 2008). As internalized materials pass from early to late endosomes and finally to lysosomes, the lumen of the endocytic organelles become more acidic. Lysosomes are the terminal compartment of both endocytic and autophagic pathways, and within lysosomes, acid hydrolase enzymes degrade proteins, lipids, and polysaccharides. The pH of the lysosome lumen is maintained between 4 and 5 (Pillay et al., 2002), which is the optimal pH for lysosomal enzyme activity.
Regulation of lysosomal pH is a complex process involving multiple channels and transporters. Acidification of lysosomes is accomplished by the electrogenic V-ATPase, which pumps protons into the lysosomal lumen (Forgac, 2007). Chloride movement through an opposite conductance pathway (Jentsch, 2007) (mediated at least in part via CLC7) and efflux of cations (Steinberg et al., 2010) facilitate vesicle acidification by neutralizing the positive charge and reducing the membrane potential caused by the pumped protons. Little is known about how the V-ATPase sets the pH or how these parallel ion transports are regulated. In particular, no pH-sensitive signaling cascades have been implicated.
Cyclic AMP has been shown to modulate lysosomal pH in macrophages (Di et al., 2006), microglia (Majumdar et al., 2007) and retinal pigment epithelium (RPE) cells (Liu et al., 2008). The cAMP effector, Protein Kinase A (PKA) increases chloride conductance (Bae and Verkman, 1990), possibly via the chloride channel CLC7. Lysosomal acidification in microglia is enhanced by upregulation of CLC7 (Majumdar et al., 2011), in what is thought to be a PKA dependent process (Majumdar et al., 2007). However, how the cAMP “second messenger” is made and whether cAMP levels are dependent upon pH remains unknown. It is tempting to postulate that sAC is the pH regulated source of cAMP regulating these processes.
Like lysosomes, both early and late endosomes are maintained within certain pH ranges; early endosomes range between pH ~5.9-6.8 whereas late endosomes range between pH ~4.9 and 6.0 (Maxfield and Yamashiro, 1987). Endosomal acidification is linked to intracellular trafficking, but it remains unknown how early endosomes “set” luminal pH to ~6.5 and late endosome/lysosomal set their luminal pH to ~5. Endosomal pH is maintained via similar proteins as control lysosomal pH, but endosomes use distinct isoforms of V-ATPases and chloride channels [for a complete review, see Forgac (2007), Stauber and Jentsch (2013)]. In such cases, different isoforms need to be trafficked to the endosomes or their activity modulated in order to establish and maintain the proper pH. sAC has already been shown to modulate the pH-dependent translocation of the V-ATPase to plasma membranes (Pastor-Soler et al., 2003; Tresguerres et al., 2010b); might sAC-generated cAMP play a role in trafficking V-ATPases or other chloride channels to endosomal/lysosomal membranes and hence establishing intra-vesicular pH?
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