A Nano-QSTR model to predict nano-cytotoxicity: an approach using human lung cells data.

BACKGROUND: The widespread use of new engineered nanomaterials (ENMs) in industries such as cosmetics, electronics, and diagnostic nanodevices, has been revolutionizing our society. However, emerging studies suggest that ENMs present potentially toxic effects on the human lung. In this regard, we developed a machine learning (ML) nano-quantitative-structure-toxicity relationship (QSTR) model to predict the potential human lung nano-cytotoxicity induced by exposure to ENMs based on metal oxide nanoparticles. RESULTS: Tree-based learning algorithms (e.g., decision tree (DT), random forest (RF), and extra-trees (ET)) were able to predict ENMs' cytotoxic risk in an efficient, robust, and interpretable way. The best-ranked ET nano-QSTR model showed excellent statistical performance with R2 and Q2-based metrics of 0.95, 0.80, and 0.79 for training, internal validation, and external validation subsets, respectively. Several nano-descriptors linked to the core-type and surface coating reactivity properties were identified as the most relevant characteristics to predict human lung nano-cytotoxicity. CONCLUSIONS: The proposed model suggests that a decrease in the ENMs diameter could significantly increase their potential ability to access lung subcellular compartments (e.g., mitochondria and nuclei), promoting strong nano-cytotoxicity and epithelial barrier dysfunction. Additionally, the presence of polyethylene glycol (PEG) as a surface coating could prevent the potential release of cytotoxic metal ions, promoting lung cytoprotection. Overall, the current work could pave the way for efficient decision-making, prediction, and mitigation of the potential occupational and environmental ENMs risks.

The system of self-consistent models based on quasi-SMILES as a tool to predict the potential of nano-inhibitors of human lung carcinoma cell line A549 for different experimental conditions.

The different features of the impact of nanoparticles on cells, such as the structure of the core, presence/absence of doping, quality of surface, diameter, and dose, were used to define quasi-SMILES, a line of symbols encoded the above physicochemical features of the impact of nanoparticles. The correlation weight for each code in the quasi-SMILES has been calculated by the Monte Carlo method. The descriptor, which is the sum of the correlation weights, is the basis for a one-variable model of the biological activity of nano-inhibitors of human lung carcinoma cell line A549. The system of models obtained by the above scheme was checked on the self-consistence, i.e., reproducing the statistical quality of these models observed for different distributions of available nanomaterials into the training and validation sets. The computational experiments confirm the excellent potential of the approach as a tool to predict the impact of nanomaterials under different experimental conditions. In conclusion, our model is a self-consistent model system that provides a user to assess the reliability of the statistical quality of the used approach.

In Vitro Cell Transformation Assays: A Valuable Approach for Carcinogenic Potentiality Assessment of Nanomaterials.

This review explores the application of in vitro cell transformation assays (CTAs) as a screening platform to assess the carcinogenic potential of nanomaterials (NMs) resulting from continuously growing industrial production and use. The widespread application of NMs in various fields has raised concerns about their potential adverse effects, necessitating safety evaluations, particularly in long-term continuous exposure scenarios. CTAs present a realistic screening platform for known and emerging NMs by examining their resemblance to the hallmark of malignancy, including high proliferation rates, loss of contact inhibition, the gain of anchorage-independent growth, cellular invasion, dysregulation of the cell cycle, apoptosis resistance, and ability to form tumors in experimental animals. Through the deliberate transformation of cells via chronic NM exposure, researchers can investigate the tumorigenic properties of NMs and the underlying mechanisms of cancer development. This article examines NM-induced cell transformation studies, focusing on identifying existing knowledge gaps. Specifically, it explores the physicochemical properties of NMs, experimental models, assays, dose and time requirements for cell transformation, and the underlying mechanisms of malignancy. Our review aims to advance understanding in this field and identify areas for further investigation.

Identifying Potential Molecular Targets in Fungi Based on (Dis)Similarities in Binding Site Architecture with Proteins of the Human Pharmacolome.

Invasive fungal infections represent a public health problem that worsens over the years with the increasing resistance to current antimycotic agents. Therefore, there is a compelling medical need of widening the antifungal drug repertoire, following different methods such as drug repositioning, identification and validation of new molecular targets and developing new inhibitors against these targets. In this work we developed a structure-based strategy for drug repositioning and new drug design, which can be applied to infectious fungi and other pathogens. Instead of applying the commonly accepted off-target criterion to discard fungal proteins with close homologues in humans, the core of our approach consists in identifying fungal proteins with active sites that are structurally similar, but preferably not identical to binding sites of proteins from the so-called "human pharmacolome". Using structural information from thousands of human protein target-inhibitor complexes, we identified dozens of proteins in fungal species of the genera Histoplasma, Candida, Cryptococcus, Aspergillus and Fusarium, which might be exploited for drug repositioning and, more importantly, also for the design of new fungus-specific inhibitors. As a case study, we present the in vitro experiments performed with a set of selected inhibitors of the human mitogen-activated protein kinases 1/2 (MEK1/2), several of which showed a marked cytotoxic activity in different fungal species.

Structural Modeling of Nanobodies: A Benchmark of State-of-the-Art Artificial Intelligence Programs.

The number of applications for nanobodies is steadily expanding, positioning these molecules as fast-growing biologic products in the biotechnology market. Several of their applications require protein engineering, which in turn would greatly benefit from having a reliable structural model of the nanobody of interest. However, as with antibodies, the structural modeling of nanobodies is still a challenge. With the rise of artificial intelligence (AI), several methods have been developed in recent years that attempt to solve the problem of protein modeling. In this study, we have compared the performance in nanobody modeling of several state-of-the-art AI-based programs, either designed for general protein modeling, such as AlphaFold2, OmegaFold, ESMFold, and Yang-Server, or specifically designed for antibody modeling, such as IgFold, and Nanonet. While all these programs performed rather well in constructing the nanobody framework and CDRs 1 and 2, modeling CDR3 still represents a big challenge. Interestingly, tailoring an AI method for antibody modeling does not necessarily translate into better results for nanobodies.

Selecting Nanobodies Specific for the Epidermal Growth Factor from a Synthetic Nanobody Library.

The epidermal growth factor (EGF) is one of the most critical ligands of the EGF receptor (EGFR), a well-known oncogene frequently overexpressed in cancerous cells and an important therapeutic target in cancer. The EGF is the target of a therapeutic vaccine aimed at inducing an anti-EGF antibody response to sequester this molecule from serum. However, strikingly, very few investigations have focused on EGF immunotargeting. Since the use of nanobodies (Nbs) for EGF neutralization may be an effective therapeutic strategy in several types of cancer, in this study, we decided to generate anti-EGF Nbs from a recently constructed, phage-displaying synthetic nanobody library. To our knowledge, this is the first attempt to obtain anti-EGF Nbs from a synthetic library. By applying a selection strategy that uses four different sequential elution steps along with three rounds of selection, we obtained four different EGF-specific Nb clones, and also tested their binding capabilities as recombinant proteins. The obtained results are very encouraging and demonstrate the feasibility of selecting nanobodies against small antigens, such as the EGF, from synthetic libraries.

Design and Construction of a Synthetic Nanobody Library: Testing Its Potential with a Single Selection Round Strategy.

Nanobodies (Nbs) are single domain antibody fragments derived from heavy-chain antibodies found in members of the Camelidae family. They have become a relevant class of biomolecules for many different applications because of several important advantages such as their small size, high solubility and stability, and low production costs. On the other hand, synthetic Nb libraries are emerging as an attractive alternative to animal immunization for the selection of antigen-specific Nbs. Here, we present the design and construction of a new synthetic nanobody library using the phage display technology, following a structure-based approach in which the three hypervariable loops were subjected to position-specific randomization schemes. The constructed library has a clonal diversity of 108 and an amino acid variability that matches the codon distribution set by design at each randomized position. We have explored the capabilities of the new library by selecting nanobodies specific for three antigens: vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) and the glycoprotein complex (GnGc) of Andes virus. To test the potential of the library to yield a variety of antigen-specific Nbs, we introduced a biopanning strategy consisting of a single selection round using stringent conditions. Using this approach, we obtained several binders for each of the target antigens. The constructed library represents a promising nanobody source for different applications.

Structural Insights into the Design of Synthetic Nanobody Libraries.

Single domain antibodies from camelids, or nanobodies, are a unique class of antibody fragments with several advantageous characteristics: small monomeric size, high stability and solubility and easy tailoring for multiple applications. Nanobodies are gaining increasing acceptance as diagnostic tools and promising therapeutic agents in cancer and other diseases. While most nanobodies are obtained from immunized animals of the camelid family, a few synthetic nanobody libraries constructed in recent years have shown the capability of generating high quality nanobodies in terms of affinity and stability. Since this synthetic approach has important advantages over the use of animals, the recent advances are indeed encouraging. Here we review over a dozen synthetic nanobody libraries reported so far and discuss the different approaches followed in their construction and validation, with an emphasis on framework and hypervariable loop design as critical issues defining their potential as high-class nanobody sources.

Key role of a structural water molecule for the specificity of 14F7-An antitumor antibody targeting the NeuGc GM3 ganglioside.

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is a monoclonal IgG1 with high clinical potential in cancer immunotherapy as it displays extraordinary specificity for NeuGc GM3, while it does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 Å crystal structure of the 14F7 antigen-binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Modeling analysis and previous mutagenesis data suggest that 14F7 may also bind to an alternative NeuGc GM3 conformation, not observed in the crystal structure. The most intriguing finding, however, was that a water molecule centrally placed in the complementarity-determining region directly mediates the specificity of 14F7 to NeuGc GM3. This has profound impact on the complexity of engineering in the binding site and provides an excellent example of the importance in understanding the water structure in antibody-antigen interactions.

Airborne particulate matter upregulates expression of early and late adhesion molecules and their receptors in a lung adenocarcinoma cell line.

BACKGROUND: Epidemiological evidence associates chronic exposure to particulate matter (PM) with respiratory damage and lung cancer. Inhaled PM may induce systemic effects including inflammation and metastasis. This study evaluated whether PM induces expression of adhesion molecules in lung cancer cells promoting interaction with monocytes. METHODS: The expression of early and late adhesion molecules and their receptors was evaluated in A549 (human lung adenocarcinoma) cells using a wide range of concentrations of PM2.5 and PM10. Then we evaluated cellular adhesion between A549 cells and U937 (human monocytes) cells after PM exposure. RESULTS: We found higher expression of both early and late adhesion molecules and their ligands in lung adenocarcinoma cells exposed to PM2.5 and PM10 particles present in the air pollution at Mexico City from 0.03 µg/cm2 with a statistically significant difference (p ≤ 0.05). PM10 had stronger effect than PM2.5. Both PM also stimulated cellular adhesion between tumor cells and monocytes. CONCLUSIONS: This study reveals a comprehensive expression profile of adhesion molecules and their ligands upregulated by PM2.5 and PM10 in A549 cells. Additionally these particles induced cellular adhesion of lung cancer cells to monocytes. This highlights possible implications of PM in two cancer hallmarks i.e. inflammation and metastasis, underlying the high cancer mortality associated with air pollution.

Genetic Modification Approaches for Parasporins Bacillus thuringiensis Proteins with Anticancer Activity.

Bacillus thuringiensis (Bt) is a bacterium capable of producing Cry toxins, which are recognized for their bio-controlling actions against insects. However, a few Bt strains encode proteins lacking insecticidal activity but showing cytotoxic activity against different cancer cell lines and low or no cytotoxicity toward normal human cells. A subset of Cry anticancer proteins, termed parasporins (PSs), has recently arisen as a potential alternative for cancer treatment. However, the molecular receptors that allow the binding of PSs to cells and their cytotoxic mechanisms of action have not been well established. Nonetheless, their selective cytotoxic activity against different types of cancer cell lines places PSs as a promising alternative treatment modality. In this review, we provide an overview of the classification, structures, mechanisms of action, and insights obtained from genetic modification approaches for PS proteins.

Understanding the disrupting mechanism of the Tau aggregation motif "306 VQIVYK311 " by phenylthiazolyl-hydrazides inhibitors.

Alzheimer's disease is a progressive neurodegenerative disorder characterized by the abnormal processing of the Tau and the amyloid precursor proteins. The unusual aggregation of Tau is based on the formation of intermolecular ß-sheets through two motifs: 275 VQIINK280 and 306 VQIVYK311 . Phenylthiazolyl-hydrazides (PTHs) are capable of inhibiting/disassembling Tau aggregates. However, the disaggregation mechanism of Tau oligomers by PTHs is still unknown. In this work, we studied the disruption of the oligomeric form of the Tau motif 306 VQIVYK311 by PTHs through molecular docking, molecular dynamics, and free energy calculations. We predicted hydrophobic interactions as the major driving forces for the stabilization of Tau oligomer, with V306 and I308 being the major contributors. Nonpolar component of the binding free energy is essential to stabilize Tau-PTH complexes. PTHs disrupted mainly the van der Waals interactions between the monomers, leading to oligomer destabilization. Destabilization of full Tau filament by PTHs and emodin was not observed in the sampled 20 ns; however, in all cases, the nonpolar component of the binding free energy is essential for the formation of Tau filament-PTH and Tau filament-emodin. These results provide useful clues for the design of more effective Tau-aggregation inhibitors.

Blocking IL-2 Signal In Vivo with an IL-2 Antagonist Reduces Tumor Growth through the Control of Regulatory T Cells.

IL-2 is critical for peripheral tolerance mediated by regulatory T (Treg) cells, which represent an obstacle for effective cancer immunotherapy. Although IL-2 is important for effector (E) T cell function, it has been hypothesized that therapies blocking IL-2 signals weaken Treg cell activity, promoting immune responses. This hypothesis has been partially tested using anti-IL-2 or anti-IL-2R Abs with antitumor effects that cannot be exclusively attributed to lack of IL-2 signaling in vivo. In this work, we pursued an alternative strategy to block IL-2 signaling in vivo, taking advantage of the trimeric structure of the IL-2R. We designed an IL-2 mutant that conserves the capacity to bind to the αß-chains of the IL-2R but not to the γc-chain, thus having a reduced signaling capacity. We show our IL-2 mutein inhibits IL-2 Treg cell-dependent differentiation and expansion. Moreover, treatment with IL-2 mutein reduces Treg cell numbers and impairs tumor growth in mice. A mathematical model was used to better understand the effect of the mutein on Treg and E T cells, suggesting suitable strategies to improve its design. Our results show that it is enough to transiently inhibit IL-2 signaling to bias E and Treg cell balance in vivo toward immunity.

Contribution of mast cells in irritant-induced airway epithelial barrier impairment in vitro.

The airway epithelium is continuously exposed to environmental irritants, which can cause adverse effects such as irritant-induced asthma (IIA). Mast cells are located near airway epithelia and are able to respond to a variety of stimuli. We aimed to investigate whether mast cells influence the response of the epithelium upon irritant exposure. Two cell lines and three different seeding conditions, that is, bronchial epithelial cells (16HBE) only, 16HBE with mast cells (HMC-1's) basolaterally, and 16HBE with HMC-1's apically, were established. Upon exposure to the environmental irritants, graphene (G), graphene oxide (GO), diesel exhaust particles (DEPs) or hypochlorite (ClO-), transepithelial electrical resistance (TEER) and paracellular flux of fluorescent-labeled dextrans were determined, along with the release of mediators. Identical experiments were conducted with the Ca2+ ionophore ionomycin. Exposure to G and GO induced a significant and permanent decrease of approximately 70% in TEER after 3 h of exposure, whereas DEP and ClO- exposure resulted in a transient decrease of approximately 20% in TEER. This response pattern was similar in all the different seeding conditions. After 24 h of exposure, fluorescein isothiocyanate-dextran transport was 10-fold greater for G and 5-fold greater for GO in each of the tested seeding conditions, while DEP and ClO- induced no change compared to the control. Upon exposure to the irritants, 16HBE did not release thymic stromal lymphopoietin, interleukin 33 (IL-33), or IL-1α, and HMC-1 cells did not release histamine, IL-6, or IL-8. Epithelial barrier integrity upon treatment with ionomycin was not affected by the presence of HMC-1 cells. A limited amount of IL-6 and IL-8 was released by ionomycin-exposed HMC-1 cells. To conclude, we found that the studied environmental irritants do not directly or indirectly activate HMC-1 cells. These mast cells did not influence the epithelial barrier function upon environmental exposure, and thus currently do not provide additional information for the underlying mechanism of IIA.

Internalization of Titanium Dioxide Nanoparticles Is Mediated by Actin-Dependent Reorganization and Clathrin- and Dynamin-Mediated Endocytosis in H9c2 Rat Cardiomyoblasts.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used for industrial and commercial applications. Once inside the body, they translocate into the bloodstream and reach different areas of the cardiovascular system including the heart, increasing the risk of developing cardiovascular diseases; consequently, the investigation of their interaction with cardiac cells is required. We previously showed that TiO2 NPs are internalized by H9c2 rat cardiomyoblasts, and here, we examined the molecular mechanisms underlying this process. TiO2 NPs internalization was evaluated by transmission electron microscopy, time-lapse microscopy, and flow cytometry. Changes in the actin cytoskeleton were studied by phalloidin staining. Endocytic uptake mechanisms for nanoparticles were probed with chemical inhibitors, whereas clathrin and dynamin expression was measured by Western blot. Cellular uptake of TiO2 NPs occurred early after 30 min exposure, and large aggregates were observed after 1 h. Actin cytoskeleton reorganization included cell elongation plus lower density and stability of actin fibers. Cytochalasin-D inhibited TiO2 NPs uptake, indicating actin-mediated internalization. Dynamin and clathrin levels increased early after TiO2 NPs exposure, and their inhibition reduced nanoparticle uptake. Therefore, TiO2 NPs internalization by H9c2 rat cardiomyoblasts involves actin cytoskeleton reorganization and clathrin/dynamin-mediated endocytosis.

An interferon gamma release assay specific for Histoplasma capsulatum to detect asymptomatic infected individuals: A proof of concept study.

Histoplasmosis is the most common endemic mycosis in the Americas. Currently, there is no laboratory test capable to detect subclinical or latent infections by Histoplasma capsulatum (Hc), which might develop as severe infections in immunocompromised individuals. For the first time to our knowledge, we explore the suitability of an interferon gamma release assay (IGRA) to detect latent Hc infection in asymptomatic individuals. A cohort of 126 volunteers was enrolled in the study, 13 of which underwent a Hc infection in the past, and 93 of them showing risk factors for this infection. The remaining 20 participants did not refer any risk factors of Hc infection, but eight of them showed evidences of infection with Mycobacterium tuberculosis. All participants were recruited in Medellin, Colombia, between January 2014 and December 2017. Whole blood samples were cultured with four different Hc crude antigens and phytohemaglutinin as positive control. The interferon (IFN)-γ released by T lymphocytes upon antigen stimulation was quantified by ELISA. A defined cutoff value of 20 pg/ml for the IFN-γ concentration allowed us to distinguish between the group with documented past infections and the group of noninfected individuals with high sensitivity (70-92%) and specificity (85-95%), for the four tested antigens. Positive 82-95% and negative 77-92% predictive values were also very high, comparable to those reported for commercially available IGRAs. The new test constitutes a promising screening method to detect individuals with latent Hc infection, even decades after the primary infection, as evidenced in this study.

Phthalate esters on urban airborne particles: Levels in PM10 and PM2.5 from Mexico City and theoretical assessment of lung exposure.

Endocrine disrupting chemicals (EDCs) from the environment are associated with reproductive abnormalities (i.e. decreased sperm concentration; increased endometriosis) and alterations of the cardiovascular system (i.e. increased blood pressure and risk of coronary disease). Some phthalates esters have been identified as EDCs, for which inhalation is considered as one of the routes of exposure. However, only little is known regarding inhalational exposure to EDCs via urban airborne particles. In the present study, we report the monthly concentration of 8 phthalate esters measured in PM10 and PM2.5 collected and recovered during 7 months in a highly populated area of Mexico City. Using the levels of PM10 and PM2.5 reported by the automatized network of environmental monitoring of Mexico City for the sampling site, we estimated exposure levels for people of different ages and gender. Two endocrine disrupting compounds, the phthalate esters DEHP and DnBP, were found on the particles in higher concentrations during the warmer months of the year. The highest concentration was reported for DEHP (229.7µg/g of particles) in PM2.5 collected in May 2013. After calculations of the DEHP concentration in the atmosphere, and using the respiratory flow rate, we determined males were potentially exposed to larger quantities of DEHP, reaching up to 18ng/8h in April 2013. Despite the concentrations of phthalates seem to be rather small, a comprehensive characterization of its presence is necessary in order to evaluate the overall exposure to these compounds, providing a clear view of exposure on children, adolescents and pregnant women.

Urban particulate matter induces the expression of receptors for early and late adhesion molecules on human monocytes.

Exposure to urban particulate matter (PM) is correlated with increases in the emergence of health services due to adverse events and deaths and is mainly related to cardiorespiratory complications. The translocation of particles from the lung into circulation has been proposed as a factor that may trigger systemic effects. Monocytes may be exposed to PM, and if the monocytes are activated, then they are likely to adhere to endothelial cells in a distant organ due to the expression of receptors for adhesion molecules. In the present study, we evaluated the expression of receptors for adhesion molecules (sLex, PSGL-1, LFA-1, VLA-4 and αVß3) in monocytes (U937 cells) exposed for 3 or 18 h to PM10 (0.001, 0.003, 0.010, 0.030, 0.300, 3 or 30 µg/mL). Exposed cells were co-cultured with human endothelial cells that were naive or previously exposed to the same particles. When U937 cells were exposed to PM10, similar levels of expression for early and late receptors for adhesion molecules were observed from 30 ng/mL as those induced by TNF-α. Cells exposed to particles at concentrations above 30 ng/mL were more adhesive to naive or exposed human endothelial cells. Taken together, our results suggest that it is plausible that activated monocytes may play a role in systemic effects induced by PM10 due to the size distribution of the particles and the concentrations required to trigger the expression of receptors for adhesion molecules in monocytes.

Internalization of Titanium Dioxide Nanoparticles Is Cytotoxic for H9c2 Rat Cardiomyoblasts.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in industry and daily life. TiO2 NPs can penetrate into the body, translocate from the lungs into the circulation and come into contact with cardiac cells. In this work, we evaluated the toxicity of TiO2 NPs on H9c2 rat cardiomyoblasts. Internalization of TiO2 NPs and their effect on cell proliferation, viability, oxidative stress and cell death were assessed, as well as cell cycle alterations. Cellular uptake of TiO2 NPs reduced metabolic activity and cell proliferation and increased oxidative stress by 19-fold measured as H2DCFDA oxidation. TiO2 NPs disrupted the plasmatic membrane integrity and decreased the mitochondrial membrane potential. These cytotoxic effects were related with changes in the distribution of cell cycle phases resulting in necrotic death and autophagy. These findings suggest that TiO2 NPs exposure represents a potential health risk, particularly in the development of cardiovascular diseases via oxidative stress and cell death.

KBE009: An antimalarial bestatin-like inhibitor of the Plasmodium falciparum M1 aminopeptidase discovered in an Ugi multicomponent reaction-derived peptidomimetic library.

Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum. Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor (Ki=0.4µM) and an in vitro antimalarial compound as potent as bestatin (IC50=18µM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC50=82µM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria.