Enhanced Immunogenicity and Protective Effects against SARS-CoV-2 Following Immunization with a Recombinant RBD-IgG Chimeric Protein.

The unprecedented global impact caused by SARS-CoV-2 imposed huge health and economic challenges, highlighting the urgent need for safe and effective vaccines. The receptor-binding domain (RBD) of SARS-CoV-2 is the major target for neutralizing antibodies and for vaccine formulations. Nonetheless, the low immunogenicity of the RBD requires the use of alternative strategies to enhance its immunological properties. Here, we evaluated the use of a subunit vaccine antigen generated after the genetic fusing of the RBD with a mouse IgG antibody. Subcutaneous administration of RBD-IgG led to the extended presence of the protein in the blood of immunized animals and enhanced RBD-specific IgG titers. Furthermore, RBD-IgG immunized mice elicited increased virus neutralizing antibody titers, measured both with pseudoviruses and with live original (Wuhan) SARS-CoV-2. Immunized K18-hACE2 mice were fully resistant to the lethal challenge of the Wuhan SARS-CoV-2, demonstrated by the control of body-weight loss and virus loads in their lungs and brains. Thus, we conclude that the genetic fusion of the RBD with an IgG molecule enhanced the immunogenicity of the antigen and the generation of virus-neutralizing antibodies, supporting the use of IgG chimeric antigens as an approach to improve the performance of SARS-CoV-2 subunit vaccines.

High Target Homology Does Not Guarantee Inhibition: Aminothiazoles Emerge as Inhibitors of Plasmodium falciparum.

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure-activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Virulence Profiles of Wild-Type, P.1 and Delta SARS-CoV-2 Variants in K18-hACE2 Transgenic Mice.

Since December 2019, the world has been experiencing the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we now face the emergence of several variants. We aimed to assess the differences between the wild-type (Wt) (Wuhan) strain and the P.1 (Gamma) and Delta variants using infected K18-hACE2 mice. The clinical manifestations, behavior, virus load, pulmonary capacity, and histopathological alterations were analyzed. The P.1-infected mice showed weight loss and more severe clinical manifestations of COVID-19 than the Wt and Delta-infected mice. The respiratory capacity was reduced in the P.1-infected mice compared to the other groups. Pulmonary histological findings demonstrated that a more aggressive disease was generated by the P.1 and Delta variants compared to the Wt strain of the virus. The quantification of the SARS-CoV-2 viral copies varied greatly among the infected mice although it was higher in P.1-infected mice on the day of death. Our data revealed that K18-hACE2 mice infected with the P.1 variant develop a more severe infectious disease than those infected with the other variants, despite the significant heterogeneity among the mice.

Protein Arginylation Is Regulated during SARS-CoV-2 Infection.

BACKGROUND: In 2019, the world witnessed the onset of an unprecedented pandemic. By February 2022, the infection by SARS-CoV-2 has already been responsible for the death of more than 5 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER stress and activation of the unfolded protein response (UPR) pathway. Degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-degron pathway. RESULTS: The role of protein arginylation during SARS-CoV-2 infection was elucidated. Protein arginylation was studied in Vero CCL-81, macrophage-like THP1, and Calu-3 cells infected at different times. A reanalysis of in vivo and in vitro public omics data combined with immunoblotting was performed to measure levels of arginyl-tRNA-protein transferase (ATE1) and its substrates. Dysregulation of the N-degron pathway was specifically identified during coronavirus infections compared to other respiratory viruses. We demonstrated that during SARS-CoV-2 infection, there is an increase in ATE1 expression in Calu-3 and Vero CCL-81 cells. On the other hand, infected macrophages showed no enzyme regulation. ATE1 and protein arginylation was variant-dependent, as shown using P1 and P2 viral variants and HEK 293T cells transfection with the spike protein and receptor-binding domains (RBD). In addition, we report that ATE1 inhibitors, tannic acid and merbromine (MER) reduce viral load. This finding was confirmed in ATE1-silenced cells. CONCLUSIONS: We demonstrate that ATE1 is increased during SARS-CoV-2 infection and its inhibition has potential therapeutic value.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin’s efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Hemorrhagic fever viruses: Pathogenesis, therapeutics, and emerging and re-emerging potential.

Hemorrhagic fever viruses (HFVs) pose a threat to global public health owing to the emergence and re-emergence of highly fatal diseases. Viral hemorrhagic fevers (VHFs) caused by these viruses are mostly characterized by an acute febrile syndrome with coagulation abnormalities and generalized hemorrhage that may lead to life-threatening organ dysfunction. Currently, the events underlying the viral pathogenicity associated with multiple organ dysfunction syndrome still underexplored. In this minireview, we address the current knowledge of the mechanisms underlying VHFs pathogenesis and discuss the available development of preventive and therapeutic options to treat these infections. Furthermore, we discuss the potential of HFVs to cause worldwide emergencies along with factors that favor their spread beyond their original niches.

Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

Targeting SUMOylation in Plasmodium as a Potential Target for Malaria Therapy.

Malaria is a parasitic disease that represents a public health problem worldwide. Protozoans of the Plasmodium genus are responsible for causing malaria in humans. Plasmodium species have a complex life cycle that requires post-translational modifications (PTMs) to control cellular activities temporally and spatially and regulate the levels of critical proteins and cellular mechanisms for maintaining an efficient infection and immune evasion. SUMOylation is a PTM formed by the covalent linkage of a small ubiquitin-like modifier protein to the lysine residues on the protein substrate. This PTM is reversible and is triggered by the sequential action of three enzymes: E1-activating, E2-conjugating, and E3 ligase. On the other end, ubiquitin-like-protein-specific proteases in yeast and sentrin-specific proteases in mammals are responsible for processing SUMO peptides and for deconjugating SUMOylated moieties. Further studies are necessary to comprehend the molecular mechanisms and cellular functions of SUMO in Plasmodium. The emergence of drug-resistant malaria parasites prompts the discovery of new targets and antimalarial drugs with novel mechanisms of action. In this scenario, the conserved biological processes regulated by SUMOylation in the malaria parasites such as gene expression regulation, oxidative stress response, ubiquitylation, and proteasome pathways, suggest PfSUMO as a new potential drug target. This mini-review focuses on the current understanding of the mechanism of action of the PfSUMO during the coordinated multi-step life cycle of Plasmodium and discusses them as attractive new target proteins for the development of parasite-specific inhibitors and therapeutic intervention toward malaria disease.

Protein corona meets freeze-drying: overcoming the challenges of colloidal stability, toxicity, and opsonin adsorption.

Freeze-drying of nanoparticle suspensions is capable of generating stable nanoformulations with improved storage times and easier transportation. Nonetheless, nanoparticle aggregation is likely induced during freeze-drying, which reduces its redispersibility upon reconstitution and leads to undesirable effects such as non-specific toxicity and impaired efficacy. In this work, bovine serum albumin (BSA) is described as a suitable protectant for silica nanoparticles (SNPs), which result in solid structures with excellent redispersibility and negligible signs of aggregation even when longer storage times are considered. We experimentally demonstrated that massive system aggregation can be prevented when a saturated BSA corona around the nanoparticle is formed before the lyophilization process. Furthermore, the BSA corona is able to suppress non-specific interactions between these nanoparticles and biological systems, as evidenced by the lack of residual cytotoxicity, hemolytic activity and opsonin adsorption. Hence, BSA can be seriously considered for industry as an additive for nanoparticle freeze-drying since it generates solid and redispersible nanoformulations with improved biocompatibility.

Insights in Chloroquine Action: Perspectives and Implications in Malaria and COVID-19.

Malaria is a threat to human mankind and kills about half a million people every year. On the other hand, COVID-19 resulted in several hundred thousand deaths since December 2019 and remains without an efficient and safe treatment. The antimalarials chloroquine (CQ) and its analog, hydroxychloroquine (HCQ), have been tested for COVID-19 treatment, and several conflicting evidence has been obtained. Therefore, the aim of this review was to summarize the evidence regarding action mechanisms of these compounds against Plasmodium and SARS-CoV-2 infection, together with cytometry applications. CQ and HCQ act on the renin angiotensin system, with possible implications on the cardiorespiratory system. In this context, flow and image cytometry emerge as powerful technologies to investigate the mechanism of therapeutic candidates, as well as for the identification of the immune response and prognostics of disease severity. Data from the large randomized trials support the conclusion that CQ and HCQ do not provide any clinical improvements in disease severity and progression of SARS-CoV-2 patients, as well as they do not present any solid evidence of increased serious side effects. These drugs are safe and effective antimalarials agents, but in SARS-CoV-2 patients, they need further studies in the context of clinical trials. © 2020 International Society for Advancement of Cytometry.

NEK10 interactome and depletion reveal new roles in mitochondria.

BACKGROUND: Members of the family of NEK protein kinases (NIMA-related kinases) were described to have crucial roles in regulating different aspects of the cell cycle. NEK10 was reported to take part in the maintenance of the G2/M checkpoint after exposure to ultraviolet light. NEK1, NEK5, NEK2 and NEK4 proteins on the other hand have been linked to mitochondrial functions. METHODS: HEK293T cells were transfected with FLAG empty vector or FLAG-NEK10 and treated or not with Zeocin. For proteomic analysis, proteins co-precipitated with the FLAG constructs were digested by trypsin, and then analyzed via LC-MS/MS. Proteomic data retrieved were next submitted to Integrated Interactome System analysis and differentially expressed proteins were attributed to Gene Ontology biological processes and assembled in protein networks by Cytoscape. For functional, cellular and molecular analyses two stable Nek10 silenced HeLa cell clones were established. RESULTS: Here, we discovered the following possible new NEK10 protein interactors, related to mitochondrial functions: SIRT3, ATAD3A, ATAD3B, and OAT. After zeocin treatment, the spectrum of mitochondrial interactors increased by the proteins: FKBP4, TXN, PFDN2, ATAD3B, MRPL12, ATP5J, DUT, YWHAE, CS, SIRT3, HSPA9, PDHB, GLUD1, DDX3X, and APEX1. We confirmed the interaction of NEK10 and GLUD1 by proximity ligation assay and confocal microscopy. Furthermore, we demonstrated that NEK10-depleted cells showed more fragmented mitochondria compared to the control cells. The knock down of NEK10 resulted further in changes in mitochondrial reactive oxygen species (ROS) levels, decreased citrate synthase activity, and culminated in inhibition of mitochondrial respiration, affecting particularly ATP-linked oxygen consumption rate and spare capacity. NEK10 depletion also decreased the ratio of mtDNA amplification, possibly due to DNA damage. However, the total mtDNA content increased, suggesting that NEK10 may be involved in the control of mtDNA content. CONCLUSIONS: Taken together these data place NEK10 as a novel regulatory player in mitochondrial homeostasis and energy metabolism.

Human Nek7-interactor RGS2 is required for mitotic spindle organization.

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.

Kinase inhibitor profile for human nek1, nek6, and nek7 and analysis of the structural basis for inhibitor specificity.

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.

Characterization of the human NEK7 interactome suggests catalytic and regulatory properties distinct from those of NEK6.

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.

"Stop Ne(c)king around": How interactomics contributes to functionally characterize Nek family kinases.

Aside from Polo and Aurora, a third but less studied kinase family involved in mitosis regulation is the never in mitosis-gene A (NIMA)-related kinases (Neks). The founding member of this family is the sole member NIMA of Aspergillus nidulans, which is crucial for the initiation of mitosis in that organism. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals: (1) centrioles/mitosis; (2) primary ciliary function/ciliopathies; and (3) DNA damage response (DDR). Recent findings, especially on Nek 1 and 8, showed however, that several Neks participate in parallel in at least two of these contexts: primary ciliary function and DDR. In the core section of this in-depth review, we report the current detailed functional knowledge on each of the 11 Neks. In the discussion, we return to the cross-connections among Neks and point out how our and other groups' functional and interactomics studies revealed that most Neks interact with protein partners associated with two if not all three of the functional contexts. We then raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize the cellular events associated with these three core functions. The new and exciting findings on the Nek family open new perspectives and should allow the Neks to finally claim the attention they deserve in the field of kinases and cell cycle biology.