Chloroquine and hydroxychloroquine in the prophylaxis and therapy of COVID-19 infection.

At the end of last century a prominent biochemist once opened the discussion of a controversial issue in the field of Bioenergetics with the following statement: "This is a long story, that shouldn't be long, but it will take a long time to make it short". As it happens, such a statement would apply perfectly well to the story of chloroquine (CQ) and hydroxychloroquine (HCQ) in the COVID-19 infection: it has become a veritable saga, with conflicting views that have often gone beyond the normal scientific dialectic, and with conclusions that have frequently been polluted by non scientific opinions: thus, for instance, when National Agencies have taken positions against CQ and HCQ, the move has been seen as a pro-vaccine attempt to block low cost therapy means. And it is difficult to avoid the feeling that the opposition to CQ and HCQ has in large measure been shaped not by scientific arguments, but by the fact that their use has been strongly endorsed by National leaders whose popularity among Western intellectuals is extremely low. The role of the two drugs in the COVID-19 infection thus deserves an objective analysis solely based on scientific facts. This contribution will attempt to produce it.

Remdesivir: From Ebola to COVID-19.

Human coronaviruses (HCoV) were discovered in the 1960s and were originally thought to cause only mild upper respiratory tract diseases in immunocompetent hosts. This view changed since the beginning of this century, with the 2002 SARS (severe acute respiratory syndrome) epidemic and the 2012 MERS (Middle East respiratory syndrome) outbreak, two zoonotic infections that resulted in mortality rates of approximately 10% and 35%, respectively. Despite the importance of these pathogens, no approved antiviral drugs for the treatment of human coronavirus infections became available. However, remdesivir, a nucleotide analogue prodrug originally developed for the treatment of Ebola virus, was found to inhibit the replication of a wide range of human and animal coronaviruses in vitro and in preclinical studies. It is therefore not surprising that when the highly pathogenic SARS-CoV-2 coronavirus emerged in late 2019 in China, causing global health concern due to the virus strong human-to-human transmission ability, remdesivir was one of the first clinical candidates that received attention. After in vitro studies had shown its antiviral activity against SARS-CoV-2, and a first patient was successfully treated with the drug in the USA, a number of trials on remdesivir were initiated. Several had encouraging results, particularly the ACTT-1 double blind, randomized, and placebo controlled trial that has shown shortening of the time to recovery in hospitalized patients treated with remdesivir. The results of other trials were instead negative. Here, we provide an overview of remdesivir discovery, molecular mechanism of action, and initial and current clinical studies on its efficacy.

History of the COVID-19 pandemic: Origin, explosion, worldwide spreading.

The SARS-CoV-2 virus of the COVID-19 pandemic, that is presently devastating the entire world, had been active well before January of this year, when its pathogenic potential exploded full force in Wuhan. It had caused the onset of small disease outbreaks in China, and probably elsewhere as well, which failed to reach epidemic potential. The distant general origin of its zoonosis can be traced back to the ecosystem changes that have decreased biodiversity, greatly facilitating the contacts between humans and the animal reservoirs that carry pathogens, including SARS-CoV-2. These reservoirs are the bats. The transition between the limited outbreaks that had occurred through 2019 and the epidemic explosion of December-January was made possible by the great amplification of the general negative conditions that had caused the preceding small outbreaks. In the light of what we have now learned, the explosion was predictable, and could have happened wherever the conditions that had allowed it, could be duplicated. What could not have been predicted was the second transition, from epidemic to pandemic. Research has now revealed that the globalization of the infection appears to have been caused by a mutation in the spike protein of the SARS-CoV-2, that has dramatically increased its transmissibility.

Biodiversity loss and COVID-19 pandemic: The role of bats in the origin and the spreading of the disease.

The loss of biodiversity in the ecosystems has created the general conditions that have favored and, in fact, made possible, the insurgence of the COVID-19 pandemic. A lot of factors have contributed to it: deforestation, changes in forest habitats, poorly regulated agricultural surfaces, mismanaged urban growth. They have altered the composition of wildlife communities, greatly increased the contacts of humans with wildlife, and altered niches that harbor pathogens, increasing their chances to come in contact with humans. Among the wildlife, bats have adapted easily to anthropized environments such as houses, barns, cultivated fields, orchards, where they found the suitable ecosystem to prosper. Bats are major hosts for αCoV and ßCoV: evolution has shaped their peculiar physiology and their immune system in a way that makes them resistant to viral pathogens that would instead successfully attack other species, including humans. In time, the coronaviruses that bats host as reservoirs have undergone recombination and other modifications that have increased their ability for inter-species transmission: one modification of particular importance has been the development of the ability to use ACE2 as a receptor in host cells. This particular development in CoVs has been responsible for the serious outbreaks in the last two decades, and for the present COVID-19 pandemic.

A V1143F mutation in the neuronal-enriched isoform 2 of the PMCA pump is linked with ataxia.

The fine regulation of intracellular calcium is fundamental for all eukaryotic cells. In neurons, Ca2+ oscillations govern the synaptic development, the release of neurotransmitters and the expression of several genes. Alterations of Ca2+ homeostasis were found to play a pivotal role in neurodegenerative progression. The maintenance of proper Ca2+ signaling in neurons demands the continuous activity of Ca2+ pumps and exchangers to guarantee physiological cytosolic concentration of the cation. The plasma membrane Ca2+ATPases (PMCA pumps) play a key role in the regulation of Ca2+ handling in selected sub-plasma membrane microdomains. Among the four basic PMCA pump isoforms existing in mammals, isoforms 2 and 3 are particularly enriched in the nervous system. In humans, genetic mutations in the PMCA2 gene in association with cadherin 23 mutations have been linked to hearing loss phenotypes, while those occurring in the PMCA3 gene were associated with X-linked congenital cerebellar ataxias. Here we describe a novel missense mutation (V1143F) in the calmodulin binding domain (CaM-BD) of the PMCA2 protein. The mutant pump was present in a patient showing congenital cerebellar ataxia but no overt signs of deafness, in line with the absence of mutations in the cadherin 23 gene. Biochemical and molecular dynamics studies on the mutated PMCA2 have revealed that the V1143F substitution alters the binding of calmodulin to the CaM-BD leading to impaired Ca2+ ejection.

Why Calcium? How Calcium Became the Best Communicator.

Calcium carries messages to virtually all important functions of cells. Although it was already active in unicellular organisms, its role became universally important after the transition to multicellular life. In this Minireview, we explore how calcium ended up in this privileged position. Most likely its unique coordination chemistry was a decisive factor as it makes its binding by complex molecules particularly easy even in the presence of large excesses of other cations, e.g. magnesium. Its free concentration within cells can thus be maintained at the very low levels demanded by the signaling function. A large cadre of proteins has evolved to bind or transport calcium. They all contribute to buffer it within cells, but a number of them also decode its message for the benefit of the target. The most important of these "calcium sensors" are the EF-hand proteins. Calcium is an ambivalent messenger. Although essential to the correct functioning of cell processes, if not carefully controlled spatially and temporally within cells, it generates variously severe cell dysfunctions, and even cell death.

The plasma membrane calcium pumps: focus on the role in (neuro)pathology.

The plasma membrane Ca2+ ATPase (PMCA pump) is a member of the superfamily of P-type pumps. It is organized in the plasma membrane with ten transmembrane helices and two main cytosolic loops, one of which contains the catalytic center. It also contains a long C-terminal tail that houses the binding site for calmodulin, the main regulator of the activity of the pump. The pump also contains a number of other regulators, among them acidic phospholipids, kinases, and numerous protein interactors. Separate genes code for 4 basic pump isoforms in mammals, additional isoform complexity being generated by the alternative splicing of primary transcripts. Pumps 1 and 4 are expressed ubiquitously, pumps 2 and 3 are tissue restricted, with preference for the nervous system. In essentially all cells, the pump coexists with much more powerful systems that clear Ca2+ from the cytosol, e.g. the SERCA pump and the Na+/Ca2+ exchanger. Its role in the global regulation of cellular Ca2+ homeostasis is thus quantitatively marginal: its main function is the regulation of Ca2+ signaling in selected sub-plasma membrane microdomains where Ca2+ modulated interactors also reside. Malfunctions of the pump linked to genetic mutations are now described with increasing frequency, the disease phenotypes being especially severe in the nervous system where isoforms 2 and 3 predominate. The analysis of the pump defects suggests that the disease phenotypes are likely to be related to the imperfect modulation of Ca2+ signaling in selected sub-plasma membrane microdomains, leading to the defective control of the activity of important Ca2+ dependent interactors.

A novel PMCA3 mutation in an ataxic patient with hypomorphic phosphomannomutase 2 (PMM2) heterozygote mutations: Biochemical characterization of the pump defect.

The neuron-restricted isoform 3 of the plasma membrane Ca2+ ATPase plays a major role in the regulation of Ca2+ homeostasis in the brain, where the precise control of Ca2+ signaling is a necessity. Several function-affecting genetic mutations in the PMCA3 pump associated to X-linked congenital cerebellar ataxias have indeed been described. Interestingly, the presence of co-occurring mutations in additional genes suggest their synergistic action in generating the neurological phenotype as digenic modulators of the role of PMCA3 in the pathologies. Here we report a novel PMCA3 mutation (G733R substitution) in the catalytic P-domain of the pump in a patient affected by non-progressive ataxia, muscular hypotonia, dysmetria and nystagmus. Biochemical studies of the pump have revealed impaired ability to control cellular Ca2+ handling both under basal and under stimulated conditions. A combined analysis by homology modeling and molecular dynamics have revealed a role for the mutated residue in maintaining the correct 3D configuration of the local structure of the pump. Mutation analysis in the patient has revealed two additional function-impairing compound heterozygous missense mutations (R123Q and G214S substitution) in phosphomannomutase 2 (PMM2), a protein that catalyzes the isomerization of mannose 6-phosphate to mannose 1-phosphate. These mutations are known to be associated with Type Ia congenital disorder of glycosylation (PMM2-CDG), the most common group of disorders of N-glycosylation. The findings highlight the association of PMCA3 mutations to cerebellar ataxia and strengthen the possibility that PMCAs act as digenic modulators in Ca2+-linked pathologies.

The ataxia related G1107D mutation of the plasma membrane Ca2+ ATPase isoform 3 affects its interplay with calmodulin and the autoinhibition process.

The plasma membrane Ca2+ ATPases (PMCA pumps) have a long, cytosolic C-terminal regulatory region where a calmodulin-binding domain (CaM-BD) is located. Under basal conditions (low Ca2+), the C-terminal tail of the pump interacts with autoinhibitory sites proximal to the active center of the enzyme. In activating conditions (i.e., high Ca2+), Ca2+-bound CaM displaces the C-terminal tail from the autoinhibitory sites, restoring activity. We have recently identified a G1107D replacement within the CaM-BD of isoform 3 of the PMCA pump in a family affected by X-linked congenital cerebellar ataxia. Here, we investigate the effects of the G1107D replacement on the interplay of the mutated CaM-BD with both CaM and the pump core, by combining computational, biochemical and functional approaches. We provide evidence that the affinity of the isolated mutated CaM-BD for CaM is significantly reduced with respect to the wild type (wt) counterpart, and that the ability of CaM to activate the pump in vitro is thus decreased. Multiscale simulations support the conclusions on the detrimental effect of the mutation, indicating reduced stability of the CaM binding. We further show that the G1107D replacement impairs the autoinhibition mechanism of the PMCA3 pump as well, as the introduction of a negative charge perturbs the contacts between the CaM-BD and the pump core. Thus, the mutation affects both the ability of the pump to optimally transport Ca2+ in the activated state, and the autoinhibition mechanism in its resting state.

A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations.

The particular importance of Ca(2+) signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca(2+) ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca(2+). A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca(2+) ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca(2+) transients generated by cell stimulation and impairs its Ca(2+) extrusion function under conditions of low resting cytosolic Ca(2+) as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca(2+)-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca(2+) homeostasis and the previous finding that PMCAs act as digenic modulators in Ca(2+)-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype.

The plasma membrane calcium pump: new ways to look at an old enzyme.

The three-dimensional structure of the PMCA pump has not been solved, but its basic mechanistic properties are known to repeat those of the other Ca(2+) pumps. However, the pump also has unique properties. They concern essentially its numerous regulatory mechanisms, the most important of which is the autoinhibition by its C-terminal tail. Other regulatory mechanisms involve protein kinases and the phospholipids of the membrane in which the pump is embedded. Permanent activation of the pump, e.g. by calmodulin, is physiologically as harmful to cells as its absence. The concept is now emerging that the global control of cell Ca(2+) may not be the main function of the pump; in some cell types, it could even be irrelevant. The main pump role would be the regulation of Ca(2+) in cell microdomains in which the pump co-segregates with partners that modulate the Ca(2+) message and transduce it to important cell functions.

Neuronal calcium signaling: function and dysfunction.

Calcium (Ca(2+)) is an universal second messenger that regulates the most important activities of all eukaryotic cells. It is of critical importance to neurons as it participates in the transmission of the depolarizing signal and contributes to synaptic activity. Neurons have thus developed extensive and intricate Ca(2+) signaling pathways to couple the Ca(2+) signal to their biochemical machinery. Ca(2+) influx into neurons occurs through plasma membrane receptors and voltage-dependent ion channels. The release of Ca(2+) from the intracellular stores, such as the endoplasmic reticulum, by intracellular channels also contributes to the elevation of cytosolic Ca(2+). Inside the cell, Ca(2+) is controlled by the buffering action of cytosolic Ca(2+)-binding proteins and by its uptake and release by mitochondria. The uptake of Ca(2+) in the mitochondrial matrix stimulates the citric acid cycle, thus enhancing ATP production and the removal of Ca(2+) from the cytosol by the ATP-driven pumps in the endoplasmic reticulum and the plasma membrane. A Na(+)/Ca(2+) exchanger in the plasma membrane also participates in the control of neuronal Ca(2+). The impaired ability of neurons to maintain an adequate energy level may impact Ca(2+) signaling: this occurs during aging and in neurodegenerative disease processes. The focus of this review is on neuronal Ca(2+) signaling and its involvement in synaptic signaling processes, neuronal energy metabolism, and neurotransmission. The contribution of altered Ca(2+) signaling in the most important neurological disorders will then be considered.

Mutation of plasma membrane Ca2+ ATPase isoform 3 in a family with X-linked congenital cerebellar ataxia impairs Ca2+ homeostasis.

Ca(2+) in neurons is vital to processes such as neurotransmission, neurotoxicity, synaptic development, and gene expression. Disruption of Ca(2+) homeostasis occurs in brain aging and in neurodegenerative disorders. Membrane transporters, among them the calmodulin (CaM)-activated plasma membrane Ca(2+) ATPases (PMCAs) that extrude Ca(2+) from the cell, play a key role in neuronal Ca(2+) homeostasis. Using X-exome sequencing we have identified a missense mutation (G1107D) in the CaM-binding domain of isoform 3 of the PMCAs in a family with X-linked congenital cerebellar ataxia. PMCA3 is highly expressed in the cerebellum, particularly in the presynaptic terminals of parallel fibers-Purkinje neurons. To study the effects of the mutation on Ca(2+) extrusion by the pump, model cells (HeLa) were cotransfected with expression plasmids encoding its mutant or wild-type (wt) variants and with the Ca(2+)-sensing probe aequorin. The mutation reduced the ability of the PMCA3 pump to control the cellular homeostasis of Ca(2+). It significantly slowed the return to baseline of the Ca(2+) transient induced by an inositol-trisphosphate (InsP(3))-linked plasma membrane agonist. It also compromised the ability of the pump to oppose the influx of Ca(2+) through the plasma membrane capacitative channels.

Amarcord: I remember.

I have tried to offer a historical account of a success story, as I saw it develop from the early times when it interested only a few aficionados to the present times when it has pervaded most of cell biochemistry and physiology. It is of course the story of calcium signaling. It became my topic of work when I was a young postdoctoral fellow at The Johns Hopkins University. I entered it through a side door, that of mitochondria, which had been my area of work during my earlier days in Italy. The 1960s and 1970s were glorious times for mitochondrial calcium signaling, but the golden period was not going to last. As I have discussed below, mitochondrial calcium gradually lost appeal, entering a long period of oblivion. Its fading happened as the general area of calcium signaling was instead experiencing a phase of explosive growth, with landmark discoveries at the molecular and cellular levels. These discoveries established that calcium signaling was one of the most important areas of cell biology. However, mitochondria as calcium partners were not dead; they were only dormant. In the 1990s, they were rescued from their state of neglect to the central position of the regulation of cellular calcium signaling, which they had once rightly occupied. Meanwhile, it had also become clear that calcium is an ambivalent messenger. Hardly anything important occurs in cells without the participation of the calcium message, but calcium must be controlled with absolute precision. This is an imperative necessity, which becomes unfortunately impaired in a number of disease conditions that transform calcium into a messenger of death.

Ca2+-activated nucleotidase 1, a novel target gene for the transcriptional repressor DREAM (downstream regulatory element antagonist modulator), is involved in protein folding and degradation.

DREAM is a Ca(2+)-dependent transcriptional repressor highly expressed in neuronal cells. A number of genes have already been identified as the target of its regulation. Targeted analysis performed on cerebella from transgenic mice expressing a dominant active DREAM mutant (daDREAM) showed a drastic reduction of the amount of transcript of Ca(2+)-activated nucleotidase 1 (CANT1), an endoplasmic reticulum (ER)-Golgi resident Ca(2+)-dependent nucleoside diphosphatase that has been suggested to have a role in glucosylation reactions related to the quality control of proteins in the ER and the Golgi apparatus. CANT1 down-regulation was also found in neuroblastoma SH-SY5Y cells stably overexpressing wild type (wt) DREAM or daDREAM, thus providing a simple cell model to investigate the protein maturation pathway. Pulse-chase experiments demonstrated that the down-regulation of CANT1 is associated with reduced protein secretion and increased degradation rates. Importantly, overexpression of wtDREAM or daDREAM augmented the expression of the EDEM1 gene, which encodes a key component of the ER-associated degradation pathway, suggesting an alternative pathway to enhanced protein degradation. Restoring CANT1 levels in neuroblastoma clones recovered the phenotype, thus confirming a key role of CANT1, and of the regulation of its gene by DREAM, in the control of protein synthesis and degradation.

Crystal structure of sarcoplasmic reticulum Ca2+-ATPase (SERCA) from bovine muscle.

The SERCA pump, a membrane protein of about 110kDa, transports two Ca(2+) ions per ATP hydrolyzed from the cytoplasm to the lumen of the sarcoplasmic reticulum. In muscle cells, its ability to remove Ca(2+) from the cytosol induces relaxation. The transport mechanism employed by the enzyme from rabbit muscle has been extensively studied, and several crystal structures representing different conformational states are available. However, no structure of the pump from other sources is known. In this paper we describe the crystal structure of the bovine enzyme, crystallized in the E1 conformation and determined at 2.9Å resolution. The overall molecular model is very similar to that of the rabbit enzyme, as expected by the high amino acid sequence identity. Nevertheless, the bovine enzyme has reduced catalytic activity with respect to the rabbit enzyme. Subtle structural modifications, in particular in the region of the long loop that protrudes into the SR lumen connecting transmembrane α-helices M7 and M8, may explain the difference.