Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability.

During the COVID-19 pandemic, Pfizer-BioNTech and Moderna successfully developed nucleoside-modified mRNA lipid nanoparticle (LNP) vaccines. SARS-CoV-2 spike protein expressed by those vaccines are identical in amino acid sequence, but several key components are distinct. Here, we compared the effect of ionizable lipids, untranslated regions (UTRs), and nucleotide composition of the two vaccines, focusing on mRNA delivery, antibody generation, and long-term stability. We found that the ionizable lipid, SM-102, in Moderna's vaccine performs better than ALC-0315 in Pfizer-BioNTech's vaccine for intramuscular delivery of mRNA and antibody production in mice and long-term stability at 4 °C. Moreover, Pfizer-BioNTech's 5' UTR and Moderna's 3' UTR outperform their counterparts in their contribution to transgene expression in mice. We further found that varying N1-methylpseudouridine content at the wobble position of mRNA has little effect on vaccine efficacy. These findings may contribute to the further improvement of nucleoside-modified mRNA-LNP vaccines and therapeutics.

cAMP-Dependent Signaling Pathways as Potential Targets for Inhibition of Plasmodium falciparum Blood Stages.

We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites.

A Fluorogenic Assay: Analysis of Chemical Modification of Lysine and Arginine to Control Proteolytic Activity of Trypsin.

The chemical modification of amino acids plays an important role in the modulation of proteins or peptides and has useful applications in the activation and stabilization of enzymes, chemical biology, shotgun proteomics, and the production of peptide-based drugs. Although chemoselective modification of amino acids such as lysine and arginine via the insertion of respective chemical moieties as citraconic anhydride and phenyl glyoxal is important for achieving desired application objectives and has been extensively reported, the extent and chemoselectivity of the chemical modification of specific amino acids using specific chemical agents (blocking or modifying agents) has yet to be sufficiently clarified owing to a lack of suitable assay methodologies. In this study, we examined the utility of a fluorogenic assay method, based on a fluorogenic tripeptide substrate (FP-AA1-AA2-AA3) and the proteolytic enzyme trypsin, in determinations of the extent and chemoselectivity of the chemical modification of lysine or arginine. As substrates, we used two fluorogenic tripeptide probes, MeRho-Lys-Gly-Leu(Ac) (lysine-specific substrate) and MeRho-Arg-Gly-Leu(Ac) (arginine-specific substrate), which were designed, synthesized, and evaluated for chemoselective modification of specific amino acids (lysine and arginine) using the fluorogenic assay. The results are summarized in terms of half-maximal inhibitory concentrations (IC50) for the extent of modification and ratios of IC50 values (IC50arginine/IC50lysine and IC50lysine/IC50arginine) as a measure of the chemoselectivity of chemical modification for amino acids lysine and arginine. This novel fluorogenic assay was found to be rapid, precise, and reproducible for determinations of the extent and chemoselectivity of chemical modification.

Bisoprolol-based 18F-PET tracer: Synthesis and preliminary in vivo validation of ß1-blocker selectivity for ß1-adrenergic receptors in the heart.

The selectivity of a drug toward various isoforms of the target protein family is important in terms of toxicology. Typically, drug or candidate selectivity is assessed by in vitro assays, but in vivo investigations are currently lacking. Positron emission tomography (PET) allows the non-invasive determination of the in vivo distribution of a radiolabeled drug, which can provide in vivo data regarding drug selectivity. Since the discovery of propranolol, a non-selective ß-blocker inhibiting both ß1- and ß2-adrenoreceptors (ß-ARs), various selective ß1-blockers, including bisoprolol, have been developed to overcome disadvantages associated with ß2-AR inhibition. As a proof of concept, we performed an in vivo PET study to understand the selectivity and efficacy of bisoprolol as a selective ß-blocker toward ß1-AR, as the heart and peripheral smooth muscles demonstrate distinct populations of ß1- and ß2-ARs. Biodistribution of 18F-labeled bisoprolol (1, [18F]bisoprolol) showed the retention of its uptake in the heart compared with other ß-AR-rich organs at late time points post-injection. The competitive blocking assay using unlabeled bisoprolol exhibited no inhibition of [18F]bisoprolol uptake in any organ but exhibited significantly rapid loss of radioactivity between two different time points in ß1-AR-rich organs such as the heart and brain. Furthermore, the organ-to-blood ratio revealed the slow excretion and better accumulation of [18F]bisoprolol inside the heart. Collectively, the ex vivo biodistribution and blocking study presented insightful evidence to better comprehend the in vivo distribution pattern of bisoprolol as a selective inhibitor targeting ß1-ARs in the heart and provided the possibility of PET as an in vivo technique for evaluating drug selectivity.

Phosphorylation-Dependent Assembly of a 14-3-3 Mediated Signaling Complex during Red Blood Cell Invasion by Plasmodium falciparum Merozoites.

Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex. Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasites.IMPORTANCE Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion. Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites.

Asymmetric and Reduced Xanthene Fluorophores: Synthesis, Photochemical Properties, and Application to Activatable Fluorescent Probes for Detection of Nitroreductase.

Xanthene fluorophores, including fluorescein, rhodol, and rhodamines, are representative classes of fluorescent probes that have been applied in the detection and visualization of biomolecules. "Turn on" activatable fluorescent probes, that can be turned on in response to enzymatic reactions, have been developed and prepared to reduce the high background signal of "always-on" fluorescent probes. However, the development of activity-based fluorescent probes for biological applications, using simple xanthene dyes, is hampered by their inefficient synthetic methods and the difficulty of chemical modifications. We have, thus, developed a highly efficient, versatile synthetic route to developing chemically more stable reduced xanthene fluorophores, based on fluorescein, rhodol, and rhodamine via continuous Pd-catalyzed cross-coupling. Their fluorescent nature was evaluated by monitoring fluorescence with variation in the concentration, pH, and solvent. As an application to activatable fluorescent probe, nitroreductase (NTR)-responsive fluorescent probes were also developed using the reduced xanthene fluorophores, and their fluorogenic properties were evaluated.

Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission.

Transmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers "rounding up" followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission.

Radiosynthesis, biological evaluation and preliminary microPET study of 18F-labeled 5-resorcinolic triazolone derivative based on ganetespib targeting HSP90.

Heat-shock protein 90 (HSP90) is a molecular chaperone that activates oncogenic transformation in several solid tumors, including lung and breast cancers. Ganetespib, a most promising candidate among several HSP90 inhibitors under clinical trials, has entered Phase III clinical trials for cancer therapy. Despite numerous evidences validating HSP90 as a target of anticancer, there are few studies on PET agents targeting oncogenic HSP90. In this study, we synthesized and biologically evaluated a novel 18F-labeled 5-resorcinolic triazolone derivative (1, [18F]PTP-Ganetespib) based on ganetespib. [18F]PTP-Ganetespib was labeled by click chemistry of Ganetespib-PEG-Alkyne (10) and [18F]PEG-N3 (11) with 37.3 ±â€¯5.11% of radiochemical yield and 99.7 ±â€¯0.09% of radiochemical purity. [18F]PTP-Ganetespib showed proper LogP (0.96 ±â€¯0.06) and good stability in human serum over 97% for 2 h. [18F]PTP-Ganetespib showed high uptakes in breast cancer cells containing triple negative breast cancer (TNBC) MDA-MB-231 and Her2-negative MCF-7 cells, which are target breast cancer cell lines of HSP90 inhibitor, ganetespib, as an anticancer. Blocking of HSP90 by the pretreatment of ganetespib exhibited significantly decreased accumulation of [18F]PTP-Ganetespib in MDA-MB-231 and MCF-7 cells, indicating the specific binding of [18F]PTP-Ganetespib to MDA-MB-231 and MCF-7 cells with high HSP90 expression. In the biodistribution and microPET imaging studies, the initial uptake into tumor was weaker than in other thoracic and abdominal organs, but [18F]PTP-Ganetespib was retained relatively longer in the tumor than other organs. The uptake of [18F]PTP-Ganetespib in tumors was not sufficient for further development as a tumor-specific PET imaging agent by itself, but this preliminary PET imaging study of [18F]PTP-Ganetespib can be basis for developing new PET imaging agents based on HSP90 inhibitor, ganetespib.

Meridianin C inhibits the growth of YD-10B human tongue cancer cells through macropinocytosis and the down-regulation of Dickkopf-related protein-3.

Meridianin C is a marine natural product known for its anti-cancer activity. At present, the anti-tumour effects of meridianin C on oral squamous cell carcinoma are unknown. Here, we investigated the effect of meridianin C on the proliferation of four different human tongue cancer cells, YD-8, YD-10B, YD-38 and HSC-3. Among the cells tested, meridianin C most strongly reduced the growth of YD-10B cells; the most aggressive and tumorigenic of the cell lines tested. Strikingly, meridianin C induced a significant accumulation of macropinosomes in the YD-10B cells; confirmed by the microscopic and TEM analysis as well as the entry of FITC-dextran, which was sensitive to the macropinocytosis inhibitor amiloride. SEM data also revealed abundant long and thin membrane extensions that resemble lamellipodia on the surface of YD-10B cells treated with meridianin C, pointing out that meridianin C-induced macropinosomes was the result of macropinocytosis. In addition, meridianin C reduced cellular levels of Dickkopf-related protein-3 (DKK-3), a known negative regulator of macropinocytosis. A role for DKK-3 in regulating macropinocytosis in the YD-10B cells was confirmed by siRNA knockdown of endogenous DKK-3, which led to a partial accumulation of vacuoles and a reduction in cell proliferation, and by exogenous DKK-3 overexpression, which resulted in a considerable inhibition of the meridianin C-induced vacuole formation and decrease in cell survival. In summary, this is the first study reporting meridianin C has novel anti-proliferative effects via macropinocytosis in the highly tumorigenic YD-10B cell line and the effects are mediated in part through down-regulation of DKK-3.

Discovery and evaluation of 3,5-disubstituted indole derivatives as Pim kinase inhibitors.

Pim kinases are promising therapeutic targets for the treatment of hematological cancers. A potent Pim kinase inhibitor 7f, derived from meridianin C, was further optimized by the replacement of 2-aminopyrimidine with substituted benzene. The optimization of the C-3 and C-5 positions of indole yielded compound 43 with improved cellular potency and high selectivity against a panel of 14 different kinases.

Acetazolamide-based [18F]-PET tracer: In vivo validation of carbonic anhydrase IX as a sole target for imaging of CA-IX expressing hypoxic solid tumors.

Carbonic anhydrase IX is overexpressed in many solid tumors including hypoxic tumors and is a potential target for cancer therapy and diagnosis. Reported imaging agents targeting CA-IX are successful mostly in clear cell renal carcinoma as SKRC-52 and no candidate was approved yet in clinical trials for imaging of CA-IX. To validate CA-IX as a valid target for imaging of hypoxic tumor, we designed and synthesized novel [18F]-PET tracer (1) based on acetazolamide which is one of the well-known CA-IX inhibitors and performed imaging study in CA-IX expressing hypoxic tumor model as 4T1 and HT-29 in vivo models other than SKRC-52. [18F]-acetazolamide (1) was found to be insufficient for the specific accumulation in CA-IX expressing tumor. This study might be useful to understand in vivo behavior of acetazolamide PET tracer and can contribute to the development of successful PET imaging agents targeting CA-IX in future. Additional study is needed to understand the mechanism of poor targeting of CA-IX, as if CA-IX is not reliable as a sole target for imaging of CA-IX expressing hypoxic solid tumors.

The central role of cAMP in regulating Plasmodium falciparum merozoite invasion of human erythrocytes.

All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K+ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca2+ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca2+ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.

Pim kinase inhibitory and antiproliferative activity of a novel series of meridianin C derivatives.

A novel series of meridianin C derivatives substituted at C-5 position were prepared. These derivatives were tested for their kinase inhibitory potencies against all three family members of the pim kinases (Pim-1, Pim-2 and Pim-3). In addition, their antiproliferative activity towards three human leukemia cell lines as MV4-11, Jurkat clone E6-1 and K562 has been evaluated. Structure activity relationships at C-3 and C-5 positions of indole were performed to better understand the mechanism behind the enhanced potency. Compound 7f, the most active compound of the series showed a single-digit nanomolar IC50 with selectivity towards Pim-1 kinase.

Role of calcineurin and actin dynamics in regulated secretion of microneme proteins in Plasmodium falciparum merozoites during erythrocyte invasion.

Plasmodium falciparum invades host erythrocytes by multiple invasion pathways. The invasion of erythrocytes by P. falciparum merozoites is a complex process that requires multiple interactions between host receptors and parasite ligands. A number of parasite proteins that mediate interaction with host receptors during invasion are localized to membrane-bound apical organelles referred to as micronemes and rhoptries. The timely release of these proteins to the merozoite surface is crucial for receptor engagement and invasion. It has been demonstrated previously that exposure of merozoites to a low potassium (K(+)) ionic environment as found in blood plasma leads to a rise in cytosolic calcium (Ca(2+)), which triggers microneme secretion. The signalling pathways that regulate microneme discharge in response to rise in cytosolic Ca(2+) are not completely understood. Here, we show that a P. falciparum Ca(2+)-dependent protein phosphatase, calcineurin (PfCN), is an essential regulator of Ca(2+)-dependent microneme exocytosis. An increase in PfCN activity was observed in merozoites following exposure to a low K(+) environment. Treatment of merozoites with calcineurin inhibitors such as FK506 and cyclosporin A prior to transfer to a low K(+) environment resulted in inhibition of secretion of microneme protein apical merozoite antigen-1 (PfAMA-1). Inhibition of PfCN was shown to result in reduced dephosphorylation and depolymerization of apical actin, which appears to be criticalfor microneme secretion. PfCN thus serves as an effector of Ca(2+)-dependent microneme exocytosis by regulating depolymerization of apical actin. Inhibitors that target PfCN block microneme exocytosis and limit growth of P. falciparum blood-stage parasites providing a novel approach towards development of new therapeutic strategies against malaria.

Suppression of LPS-induced inflammatory activities by Rosmarinus officinalis L.

Rosemary (Rosmarinus officinalis L.) has been used in folk medicine to treat headaches, epilepsy, poor circulation, and many other ailments. It was found that rosemary could act as a stimulant and mild analgesic and could reduce inflammation. However, the mechanisms underlying the anti-inflammatory effects of rosemary need more study to be established. Therefore, in this study, the effects of rosemary on the activation of nuclear factor kappa beta (NF-kB) and mitogen-activated protein kinases (MAPKs), the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and cytokine in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were investigated. A methanol extract of rosemary and its hexane fraction reduced NO generation with an IC(50) of 2.75 and 2.83 µg/ml, respectively. Also, the methanol extract and the hexane fraction inhibited LPS-induced MAPKs and NF-kB activation associated with the inhibition of iNOS or COX-2 expression. LPS-induced production of PGE(2) and tumour necrosis factor-alpha (TNF-α) were blocked by rosemary. Rosemary extract and its hexane fraction are important for the prevention of phosphorylation of MAPKs, thereby blocking NF-kB activation, which in turn leads to decreased expression of iNOS and COX-2, thus preventing inflammation.

Characterization of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) and its role in microneme secretion during erythrocyte invasion.

Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu(356) to Thr(370)) of the JD in the presence of calcium (Ca(2+)). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.

Identification of a potent combination of key Plasmodium falciparum merozoite antigens that elicit strain-transcending parasite-neutralizing antibodies.

Blood-stage malaria vaccines that target single Plasmodium falciparum antigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood-stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain-transcending, invasion-inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently. Our approach is to develop a receptor-blocking blood-stage vaccine against P. falciparum that targets the erythrocyte binding domains of multiple parasite adhesins, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition. We evaluated the invasion-inhibitory activities of 20 different triple combinations of antibodies mixed in vitro against a diverse set of six key merozoite ligands, including the novel ligands P. falciparum apical asparagine-rich protein (PfAARP), EBA-175 (PfF2), P. falciparum reticulocyte binding-like homologous protein 1 (PfRH1), PfRH2, PfRH4, and Plasmodium thrombospondin apical merozoite protein (PTRAMP), which are localized in different apical organelles and are translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination of EBA-175 (PfF2), PfRH2, and PfAARP produced the most efficacious strain-transcending inhibition of erythrocyte invasion against diverse P. falciparum clones. This potent antigen combination was selected for coimmunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. We have thus demonstrated a novel two-step screening approach to identify a potent antigen combination that elicits strong strain-transcending invasion inhibition, supporting its development as a receptor-blocking malaria vaccine.