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Antimalarial drugs are an urgently need and crucial tool in the campaign against malaria, which can threaten public health. In this study, we examined the cytotoxicity of the 9 antimalarial compounds chemically synthesized using SKM13-2HCl. Except for SKM13-2HCl, the 5 newly synthesized compounds had a 50% cytotoxic concentration (CC50) > 100 µM, indicating that they would be less cytotoxic than SKM13-2HCl. Among the 5 compounds, only SAM13-2HCl outperformed SKM13-2HCl for antimalarial activity, showing a 3- and 1.3-fold greater selective index (SI) (CC50/IC50) than SKM13-2HCl in vitro against both chloroquine-sensitive (3D7) and chloroquine -resistant (K1) Plasmodium falciparum strains, respectively. Thus, the presence of morpholine amide may help to effectively suppress human-infectious P. falciparum parasites. However, the antimalarial activity of SAM13-2HCl was inferior to that of the SKM13-2HCl template compound in the P. berghei NK65-infected mouse model, possibly because SAM13-2HCl had a lower polarity and less efficient pharmacokinetics than SKM13-2HCl. SAM13-2HCl was more toxic in the rodent model. Consequently, SAM13-2HCl containing morpholine was selected from screening a combination of pharmacologically significant structures as being the most effective in vitro against human-infectious P. falciparum but was less efficient in vivo in a P. berghei-infected animal model when compared with SKM13-2HCl. Therefore, SAM13-2HCl containing morpholine could be considered a promising compound to treat chloroquine-resistant P. falciparum infections, although further optimization is crucial to maintain antimalarial activity while reducing toxicity in animals.
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Antimaláricos , Antagonistas do Ácido Fólico , Camundongos , Animais , Humanos , Antimaláricos/farmacologia , Camundongos Endogâmicos ICR , Plasmodium berghei , Plasmodium falciparum , Cloroquina/farmacologia , Morfolinas , Amidas/farmacologia , Modelos Animais de DoençasRESUMO
In this study, an intense pulsed light (IPL) annealing process for a printed multi-layered indium-gallium-zinc-oxide (IGZO) and silver (Ag) electrode structure was developed for a high performance all-printed inorganic thin film transistor (TFT). Through a solution process using IGZO precursor and Ag ink, the bottom gate structure TFT was fabricated. The spin coating method was used to form the IGZO semiconductor layer on a heavily-doped silicon wafer covered with thermally grown silicon dioxide. The annealing process of the IGZO layer utilized an optimized IPL irradiation process. The Ag inks were printed on the IGZO layer by screen printing to form the source and drain (S/D) pattern. This S/D pattern was dried by near infrared radiation (NIR) and the dried S/D pattern was sintered with intense pulsed light by varying the irradiation energy. The performances of the all-printed TFT such as the field effect mobility and on-off ratio electrical transfer properties were measured by a parameter analyzer. The interfacial analysis including the contact resistance and cross-sectional microstructure analysis is essential because diffusion phenomenon can occur during the annealing and sintering process. Consequently, this TFT device showed noteworthy performance (field effect mobility: 7.96 cm2/V s, on/off ratio: 107). This is similar performance compared to a conventional TFT, which is expected to open a new path in the printed metal oxide-based TFT field.
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A stimuli-responsive protein self-assembly offers promising utility as a protein nanocage for biotechnological and medical applications. Herein, the development of a virus-like particle (VLP) that undergoes a transition between assembly and disassembly under a neutral and acidic pH, respectively, for a targeted delivery is reported. The structure of the bacteriophage P22 coat protein is used for the computational design of coat subunits that self-assemble into a pH-responsive VLP. Subunit designs are generated through iterative computational cycles of histidine substitutions and evaluation of the interaction energies among the subunits under an acidic and neutral pH. The top subunit designs are tested and one that is assembled into a VLP showing the highest pH-dependent structural transition is selected. The cryo-EM structure of the VLP is determined, and the structural basis of a pH-triggered disassembly is delineated. The utility of the designed VLP is exemplified through the targeted delivery of a cytotoxic protein cargo into tumor cells in a pH-dependent manner. These results provide strategies for the development of self-assembling protein architectures with new functionality for diverse applications.
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Bacteriófago P22 , Proteínas do Capsídeo , Proteínas do Capsídeo/metabolismo , Bacteriófago P22/química , Bacteriófago P22/metabolismo , Concentração de Íons de HidrogênioRESUMO
Intrinsically disordered proteins (IDPs) not only play important roles in biological processes but are also linked with the pathogenesis of various human diseases. Specific and reliable sensing of IDPs is crucial for exploring their roles but remains elusive due to structural plasticity. Here, we present the development of a new type of fluorescent protein for the ratiometric sensing and tracking of an IDP. A ß-strand of green fluorescent protein (GFP) was truncated, and the resulting GFP was further engineered to undergo the transition in the absorption maximum upon binding of a target motif within amyloid-ß (Aß) as a model IDP through rational design and directed evolution. Spectroscopic and structural analyses of the engineered truncated GFP demonstrated that a shift in the absorption maximum is driven by the change in the chromophore state from an anionic (460 nm) state into a neutral (390 nm) state as the Aß binds, allowing a ratiometric detection of Aß. The utility of the developed GFP was shown by the efficient and specific detection of an Aß and the tracking of its conformational change and localization in astrocytes.
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Coronavirus Disease 2019 (COVID-19) pandemic is severely impacting the world, and tremendous efforts have been made to deal with it. Despite many advances in vaccines and therapeutics, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains an intractable challenge. We present a bivalent Receptor Binding Domain (RBD)-specific synthetic antibody, specific for the RBD of wild-type (lineage A), developed from a non-antibody protein scaffold composed of LRR (Leucine-rich repeat) modules through phage display. We further reinforced the unique feature of the synthetic antibody by constructing a tandem dimeric form. The resulting bivalent form showed a broader neutralizing activity against the variants. The in vivo neutralizing efficacy of the bivalent synthetic antibody was confirmed using a human ACE2-expressing mouse model that significantly alleviated viral titer and lung infection. The present approach can be used to develop a synthetic antibody showing a broader neutralizing activity against a multitude of SARS-CoV-2 variants.
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COVID-19 , SARS-CoV-2 , Animais , Camundongos , Humanos , SARS-CoV-2/genética , Anticorpos , Técnicas de Visualização da Superfície Celular , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêuticoRESUMO
Bilateral vertebral artery occlusive disease has been considered as a favorable condition with good collaterals. However, the prognosis of acute ischemic stroke secondary to symptomatic bilateral vertebral artery occlusion (BVAO) and endovascular treatment (EVT) has rarely been reported. We retrospectively selected patients with acute ischemic stroke admitted for symptomatic BVAO between January 2020 and February 2023. All patients with ischemic stroke were evaluated for ischemic lesion and arterial status using brain imaging and angiography. The prognosis of acute stroke with symptomatic BVAO was compared between EVT and conventional treatment. Outcomes were evaluated using modified Rankin Scale (mRS) score at 3 months follow-up. Within the study period, 17 of 2,655 acute ischemic stroke patients were diagnosed with ischemic stroke with symptomatic BVAO. The median age of these patients was 70 (interquartile range 44-89) years, and 13 (76%) were male. Seven patients received emergent EVT with stenting and 10 patients received conventional medical treatment only. Nine of 10 patients with conventional treatment had in-hospital stroke progression and developed new ischemic lesions in the pons and midbrain. Five patients with fetal and hypoplastic posterior communicating artery presented bilateral cerebral peduncular lesions. At 3 months follow-up, 6 patients (35%) had favorable outcomes (mRS 0-2), of which 5 were treated with vertebral artery stenting and 1 received conventional treatment. Ischemic stroke in patients with acute symptomatic BVAO is uncommon. However, stroke progression is common, and the prognosis of most patients is poor. Rescue management such as EVT might be considered for symptomatic BVAO.
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PURPOSE: Patients with minor stroke (National Institutes of Health Stroke Scale score ≤5) and large vessel occlusion (LVO) often experience neurological deterioration >24 hours after onset. However, the efficacy of endovascular reperfusion therapy in these patients remains unclear. The aim of this study was to determine the efficacy and safety of reperfusion therapy in patients with minor stroke and neurological deterioration >24 hours after onset. MATERIALS AND METHODS: Data were retrospectively reviewed from patients between January 2019 and April 2022 who met the following criteria: (1) minor stroke and small definitive ischemic lesions at initial visit, (2) onset to neurological deterioration >24 hours, (3) cortical signs, Alberta Stroke Program Early computed tomography (CT) Score >6 points, and large artery occlusion confirmed by CT angiography at neurological deterioration. Efficacy and safety outcomes were based on final thrombolysis in cerebral infarction (TICI), incidence of symptomatic intracranial hemorrhage (ICH), and mortality. Outcomes were assessed using the modified Rankin Scale (mRS) at 3 months. Good outcome was defined as a mRS of 0, 1, or 2. RESULTS: Data from 26 patients (38.4% female, mean age 75.8 years) were analyzed; 18 (69.2%) had a good outcome. A final TICI of 2b or 3 was observed in 24 (92.3%) patients. No other adverse events, including dissection, vasospasm or distal embolization, were observed during the procedures. Hemorrhagic events occurred in 8 patients after the procedure; however, there were no symptomatic ICHs. Good prognostic factors were younger age (P=0.062) and carotid stenting (P=0.025). CONCLUSION: Endovascular reperfusion therapy performed in selected patients with minor stroke, LVO, and neurological deterioration >24 hours after stroke onset demonstrated favorable outcomes and safety.
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Inflammasomes are multi-protein complexes and play a crucial role in host defense against pathogens. Downstream inflammatory responses through inflammasomes are known to be related to the oligomerization degree of ASC specks, but the detailed mechanism still remains unexplored. Here, we demonstrate that oligomerization degrees of ASC specks regulate the caspase-1 activation in the extracellular space. A protein binder specific for a pyrin domain (PYD) of ASC (ASCPYD) was developed, and structural analysis revealed that the protein binder effectively inhibits the interaction between PYDs, disassembling ASC specks into low oligomeric states. ASC specks with a low oligomerization degree were shown to enhance the activation of caspase-1 by recruiting and processing more premature caspase-1 through interactions between CARD of caspase-1 (caspase-1CARD) and CARD of ASC (ASCCARD). These findings can provide insight into controlling the inflammasome-mediated inflammatory process as well as the development of inflammasome-targeting drugs.
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Small GTPases are key signaling nodes that regulate the cellular processes and subcellular events, and their abnormal activities and dysregulations are closely linked with diverse cancers. Here, we report the development of conformation-selective protein binders for a KRAS mutant. The conformation-specific protein binders were selected from a repebody scaffold composed of LRR (Leucine-rich repeat) modules through phage display and modular engineering against constitute active conformation of KRAS. Epitope of the selected binders was mapped to be located close to switch I of KRAS. The conformation-selective protein binders were shown to effectively block the interaction between active KRAS and RAS-binding domain of BRAF, suppressing the KRAS-mediated downstream signaling.
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Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Domínios Proteicos , MutaçãoRESUMO
Protein assemblies have drawn much attention as platforms for biomedical applications, including gene/drug delivery and vaccine, due to biocompatibility and functional diversity. Here, the construction and functionalization of a protein assembly composed of human clathrin heavy chain and light chain for a targeted protein delivery, is presented. The clathrin heavy and light chains are redesigned and associated with each other, and the resulting triskelion unit further self-assembled into a clathrin assembly with the size of about 28 nm in diameter. The clathrin assembly is dual-functionalized with a protein cargo and a targeting moiety using two different orthogonal protein-ligand pairs through one-pot reaction. The functionalized clathrin assembly exhibits about a 900-fold decreased KD value for a cell-surface target due to avidity compared to a native targeting moiety. The utility of the clathrin assembly is demonstrated by an efficient delivery of a protein cargo into tumor cells in a target-specific manner, resulting in a strong cytotoxic effect. The present approach can be used in the creation of protein assemblies with multimodality.
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Clatrina , Sistemas de Liberação de Medicamentos , Humanos , Clatrina/metabolismoRESUMO
Malaria is among the most devastating and widespread tropical parasitic diseases in developing countries. To prevent a potential public health emergency, there is an urgent need for new antimalarial drugs, with single-dose cures, broad therapeutic potential, and novel mechanism of action. We synthesized HCl salt of SKM13 (SKM13-2HCl) based on the modification of SKM13 to improve solubility in water. The anti-malarial activity of the synthesized drug was evaluated in both in vitro and in vivo models. The selective index indicated that SKM13-2HCl showed the same effectiveness with SKM13 in Plasmodium falciparum in in-vitro. Even though, in vivo mouse study demonstrated that SKM13 (20 mg/kg) at single dose could not completely inhibit P. berghei growth in blood. The survival rate increased from 33 to 90% at 15 days after infection. However, SKM13-2HCl (20 mg/kg) at a single dose increased the survival rate up to 100% at the same duration. Ultra-High-Performance Liquid Chromatography (UHPLC) showed that solubility in water of SKM13 and SKM13-2HCL was 0.389 mg/mL and 417 mg/mL, respectively. Pharmacokinetics (PK) analysis corresponded to the increased solubility of SKM13-2HCl over SKM13. Haematological parameters [red blood cell (RBC) count, haemoglobin level, and haematocrit level] supported the comparable efficacy of SKM13 and SKM13-2HCl in a 4-day suppression test. One mode of these drugs was found to be activating phosphorylation of eIF2α, hallmark of ER-stress, to kill parasite. Novel salt derivative of SKM13 (SKM13-2HCl) have enhanced anti-malarial activity against P. falciparum with endoplasmic reticulum (ER)-stress and salt form of SKM13 is an excellent direction to develop anti-malarial drug candidate in mice model.
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Antimaláricos , Malária Falciparum , Malária , Camundongos , Animais , Antimaláricos/uso terapêutico , Plasmodium berghei , Plasmodium falciparum , Malária/tratamento farmacológico , Malária/parasitologia , Malária Falciparum/tratamento farmacológico , ÁguaRESUMO
Fever is a typical symptom of most infectious diseases. While prolonged fever may be clinically undesirable, mild reversible fever (<39â, 312 K) can potentiate the immune responses against pathogens. Here, using molecular dynamics and free energy calculations, we investigated the effect of febrile temperatures (38â to 40â, 311 K to 313 K) on the immune complexes formed by the SARS-CoV-2 spike protein with two neutralizing monoclonal antibodies. In analyzing the conformational dynamics of the interactions between the antibodies and the spike protein under different thermal conditions, we found that, at mild fever temperatures (311-312 K), the binding affinities of the two antibodies improve when compared to the physiological body temperature (37â, 310 K). Furthermore, only at 312 K, antibodies exert distinct mechanical effects on the receptor binding domains of the spike protein that may hinder SARS-CoV-2 infectivity. Enhanced antibody binding affinity may thus be obtained using appropriate temperature conditions.
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Globally, point-of-care testing (POCT) is the most preferable on-site technique for disease detection and includes a rapid diagnostic test (RDT) and fluorescent immunochromatographic strip test (FICT). The testing kits are generally insufficient in terms of signal enhancement, which is a major drawback of this approach. Sensitive and timely on-site POCT methods with high signal enhancement are therefore essential for the accurate diagnosis of infectious diseases. Herein, we prepare cysteamine-gold coated carboxylated europium chelated nanoparticle (Cys Au-EuNPs)-mediated POCT for the detection of the H5N1 avian influenza virus (AIV). Commercial nanoparticles were used for comparison. The spectral characteristics, surface morphologies, functional groups, surface charge and stability of the Cys AuNPs, EuNPs, and Cys Au-EuNPs were confirmed by UV-visible spectrophotometry, fluorescence spectrometry, transmission electron microscope with Selected area electron diffraction (TEM-SAED), Fourier-transform infrared spectroscopy (FTIR) and zeta potential analysis. The particle size distribution revealed an average size of ~130 ± 0.66 nm for the Cys Au-EuNPs. The Cys Au-EuNP-mediated RDT (colorimetric analysis) and FICT kit revealed a limit of detection (LOD) of 10 HAU/mL and 2.5 HAU/mL, respectively, for H5N1 under different titer conditions. The obtained LOD is eight-fold that of commercial nanoparticle conjugates. The photo luminance (PL) stability of ~3% the Cys Au-EuNPs conjugates that was obtained under UV light irradiation differs considerably from that of the commercial nanoparticle conjugates. Overall, the developed Cys Au-EuNPs-mediated dual-mode POCT kit can be used as an effective nanocomposite for the development of on-site monitoring systems for infectious disease surveillance.
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Virus da Influenza A Subtipo H5N1 , Nanopartículas Metálicas , Animais , Cisteamina , Ouro/química , Nanopartículas Metálicas/química , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Human interleukin-15 (hIL-15) has attracted a considerable attention as a promising cancer immunotherapeutic due to its function to directly stimulate the proliferation and cytotoxic activity of NK and T cells. Nevertheless, a relatively short half-life of hIL-15 requires repeated administration and higher doses, causing serious side effects. Here, we demonstrate an enhanced blood half-life and biological activity of hIL-15 through genetic fusion of a human serum albumin-specific protein binder (rHSA). The fusion construct (rHSA-IL15) was observed to maintain respective binding activities for both hIL-15 receptor α and human serum albumin. The rHSA-IL15 led to a significant increase in the secretion of Granzyme B and INF-γ by immune cells compare to free hIL-15, expanding the population of activated T cell subset such as CD4 + T and CD8+ T cells. The terminal half-life of the rHSA-IL15 was prolonged by around a 40-fold in transgenic mice expressing human serum albumin, compared to free hIL-15. The rHSA-IL15 resulted in distinct anti-tumor activities in xenograft SCC (squamous cell carcinoma) mouse and allograft melanoma mouse models through activation of NK and CD8+ T cells. The rHSA-IL15 is expected to be used in cancer immunotherapy, assisting in the development of other cytokines as immunotherapeutic agents with greater efficacy.
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Interleucina-15 , Neoplasias , Animais , Linfócitos T CD8-Positivos , Proliferação de Células , Meia-Vida , Humanos , Interleucina-15/genética , Interleucina-15/metabolismo , Interleucina-2 , Camundongos , Neoplasias/tratamento farmacológico , Albumina Sérica , Albumina Sérica Humana/farmacologiaRESUMO
The therapeutic efficacy of a protein binder largely depends on two factors: its binding site and its binding affinity. Advances in in vitro library display screening and next-generation sequencing have enabled accelerated development of strong binders, yet identifying their binding sites still remains a major challenge. The differentiation, or "binning", of binders into different groups that recognize distinct binding sites on their target is a promising approach that facilitates high-throughput screening of binders that may show different biological activity. Here we study the extent to which the information contained in the amino acid sequences comprising a set of target-specific binders can be leveraged to bin them, inferring functional equivalence of their binding regions, or paratopes, based directly on comparison of the sequences, their modeled structures, or their modeled interactions. Using a leucine-rich repeat binding scaffold known as a "repebody" as the source of diversity in recognition against interleukin-6 (IL-6), we show that the "Epibin" approach introduced here effectively utilized structural modelling and docking to extract specificity information encoded in the repebody amino acid sequences and thereby successfully recapitulate IL-6 binding competition observed in immunoassays. Furthermore, our computational binning provided a basis for designing in vitro mutagenesis experiments to pinpoint specificity-determining residues. Finally, we demonstrate that the Epibin approach can extend to antibodies, retrospectively comparing its predictions to results from antigen-specific antibody competition studies. The study thus demonstrates the utility of modeling structure and binding from the amino acid sequences of different binders against the same target, and paves the way for larger-scale binning and analysis of entire repertoires.
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Acute internal carotid artery (ICA) occlusions cause extensive brain ischemia. Accurate determination of the occlusion site facilitates rapid revascularization interventions and improves prognosis. However, proximal ICA occlusions, as determined with computed tomography (CT) angiography, often are located more distally. Therefore, we assessed clinical and imaging factors associated with the accurate determination of occlusion sites. In this observational study, we evaluated 102 patients who presented acute ischemic stroke symptoms and had a CT angiography within 6 h, showing proximal ICA occlusion. The participants were divided into two groups, depending on whether there was correspondence between digital subtraction angiography and CT angiography regarding the occlusion location. Proximal occlusions were, accordingly, categorized as "true" (correspondence) or "false" (no correspondence; distal). Demographic, clinical, and imaging features were analyzed. Multivariate regression analysis was performed to identify factors predicting the correspondence between actual ICA occlusion sites and those detected by CT angiography. The shape (Odds ratios, OR = 646.584; Confidence interval, CI = 21.703-19263.187; p < 0.001) and the length (OR = 0.696; CI = 0.535-0.904; p = 0.007) of the ICA occlusion and atrial fibrillation (OR = 0.024; CI = 0.002-0.340; p = 0.006) were significant factors. The cut-off length of ICA stump at 6.2 mm, the sensitivity was 71%, and the specificity was 70% (area under the ROC curve = 0.767).
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Tension pneumocephalus is a neurosurgical emergency that occurs when air is trapped in the intracranial cavity, leading to brain compression and causing severe neurological symptoms such as decreases in motor function, sensory function, and consciousness. Most cases of pneumocephalus require conservative treatment; however, because of the possible fatal complications, rapid diagnosis and appropriate treatment are important. Here, we present the case of an 81-year-old male patient who had undergone head trauma three hours prior to being admitted to our emergency room (ER) because of mental cloudiness. The radiologic findings showed tension pneumocephalus caused by an ethmoidal roof fracture. Emergency reconstruction of the ethmoidal roof with craniotomy was performed to remove the intracranial air using normal saline irrigation. By sharing our experience with this case, we hope to provide an option for the treatment of such cases.
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Antimalarial drugs play an important role in the control and treatment of malaria, a deadly disease caused by the protozoan parasite Plasmodium spp. The development of novel antimalarial agents effective against drug-resistant malarial parasites is urgently needed. The novel derivatives, SKM13-MeO and SKM13-F, were designed based on an SKM13 template by replacing the phenyl group with electron-donating (-OMe) or electron-withdrawing groups (-F), respectively, to reverse the electron density. A colorimetric assay was used to quantify cytotoxicity, and in vitro inhibition assays were performed on 3 different blood stages (ring, trophozoite, and schizonts) of P. falciparum 3D7 and the ring/mixed stage of D6 strain after synchronization. The in vitro cytotoxicity analysis showed that 2 new SKM13 derivatives reduced the cytotoxicity of the SKM13 template. SKM13 maintained the IC50 at the ring and trophozoite stages but not at the schizont stage. The IC50 values for both the trophozoite stage of P. falciparum 3D7 and ring/mixed stages of D6 demonstrated that 2 SKM13 derivatives had decreased antimalarial efficacy, particularly for the SKM13-F derivative. SKM13 may be comparably effective in ring and trophozoite, and electron-donating groups (-OMe) may be better maintain the antimalarial activity than electron-withdrawing groups (-F) in SKM13 modification.
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Antimaláricos , Malária Falciparum , Malária , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Plasmodium falciparum , Malária Falciparum/tratamento farmacológico , TrofozoítosRESUMO
The assembly of proteins in a programmable manner provides insight into the creation of novel functional nanomaterials for practical applications. Despite many advances, however, a rational protein assembly with an easy scalability in terms of size and valency remains a challenge. Here, a simple bottom-up approach to the supramolecular protein assembly with a tunable size and valency in a programmable manner is presented. The dendrimer-like protein assembly, simply called a "protein dendrimer," is constructed through a stepwise and alternate addition of a building block protein. Starting from zeroth-generation protein dendrimer (pG0 ) of 27 kDa, the protein dendrimer is sequentially grown to pG1 , pG2 , pG3 , to pG4 with a molecular mass of 94, 216, 483, and 959 kDa, respectively. The valency of the protein dendrimers at the periphery increases by a factor of two after each generation, allowing a tunable valency and easy functionalization. The protein dendrimers functionalizes with a targeting moiety and a cytotoxic protein cargo shows a typical feature of multi-valency in the avidity and a highly enhanced cellular cytotoxicity, exemplifying their utility as a protein delivery platform. The present approach can be effectively used in the creation of protein architectures with new functions for biotechnological and medical applications.