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Emicizumab improves the procoagulant activity of select loss-of-function FIX variants with likely dysfunctional assembly of the intrinsic Xase complex that cause hemophilia B. FVIII-mimetics may represent an alternative non-factor therapy for select hemophilia B patients.
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Durable factor VIII (FVIII) expression that normalizes hemostasis is an unrealized goal of hemophilia A adeno-associated virus (AAV)-mediated gene therapy. Trials with initial normal FVIII activity observed unexplained year-over-year declines in expression while others reported low-level, stable FVIII expression inadequate to restore normal hemostasis. Here we demonstrate that mice recapitulate FVIII expression-level-dependent loss of plasma FVIII levels due to declines in vector copy number. We show that an enhanced function FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ), resistant to inactivation by protein C, normalizes hemostasis at below-normal expression levels without evidence of prothrombotic risk in hemophilia A mice. These data support that FVIII-QQ may restore normal FVIII function at low-levels of expression to permit durability using low AAV vector doses to minimize dose-dependent AAV toxicities. This work informs the mechanism of FVIII durability after AAV gene transfer and supports that incorporating the FVIII-QQ transgene may safely overcome current hemophilia A gene therapy limitations.
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Drug resistance is a constant threat to malaria control efforts making it important to maintain a good pipeline of new drug candidates. Of particular need are compounds that also block transmission by targeting sexual stage parasites. Mature sexual stages are relatively resistant to all currently used antimalarials except the 8-aminoquinolines that are not commonly used due to potential side effects. Here, we synthesized a new Torin 2 derivative, NCATS-SM3710 with increased aqueous solubility and specificity for Plasmodium and demonstrate potent in vivo activity against all P. berghei life cycle stages. NCATS-SM3710 also has low nanomolar EC50s against in vitro cultured asexual P. falciparum parasites (0.38 ± 0.04 nM) and late stage gametocytes (5.77 ± 1 nM). Two independent NCATS-SM3710/Torin 2 resistant P. falciparum parasite lines produced by growth in sublethal Torin 2 concentrations both had genetic changes in PF3D7_0509800, annotated as a phosphatidylinositol 4 kinase (Pfâ¯PI4KIIIß). One line had a point mutation in the putative active site (V1357G), and the other line had a duplication of a locus containing Pfâ¯PI4KIIIß. Both lines were also resistant to other Pfâ¯PI4K inhibitors. In addition NCATS-SM3710 inhibited purified Pfâ¯PI4KIIIß with an IC50 of 2.0 ± 0.30 nM. Together the results demonstrate that Pfâ¯PI4KIIIß is the target of Torin 2 and NCATS-SM3710 and provide new options for potent multistage drug development.
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Recently, we heterologously expressed, purified, and analyzed the function of the sole Plasmodium falciparum phosphatidylinositol 3-kinase (PI3K), found that the enzyme is a "class III" or "Vps34" PI3K, and found that it is irreversibly inhibited by Fe2+-mediated covalent, nonspecific interactions with the leading antimalarial drug, dihydroartemisinin [Hassett, M. R., et al. (2017) Biochemistry 56, 4335-4345]. One of several P. falciparum phosphatidylinositol 4-kinases [putative IIIß isoform (PfPI4KIIIß)] has generated similar interest as a druggable target; however, no validation of the mechanism of action for putative PfPI4K inhibitors has yet been possible due to the lack of purified PfPI4KIIIß. We therefore codon optimized the pfpi4kIIIß gene, successfully expressed the protein in yeast, and purified an N-lobe catalytic domain PfPI4KIIIß protein. Using an enzyme-linked immunosorbent assay strategy previously perfected for analysis of PfPI3K (PfVps34), we measured the apparent initial rate, Km,app(ATP), and other enzyme characteristics and found full activity for the construct and that PfPI4KIIIß activity is most consistent with the class IIIß designation. Because several novel antimalarial drug candidates with different chemical scaffolds have been proposed to target PfPI4KIIIß, we titrated enzyme inhibition for these candidates versus purified PfPI4KIIIß and PfVps34. We also analyzed the activity versus purified PfPI4KIIIß mutants previously expressed in P. falciparum selected for resistance to these drugs. Interestingly, we found that a putative PfPI4KIIIß inhibitor currently in advanced trials (MMV390048; MMV '0048) is a potent inhibitor of both PfVps34 and PfPI4KIIIß. These data are helpful for further preclinical optimization of an exciting new class of P. falciparum PI kinase inhibitor ("PfPIKi") antimalarial drugs.
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1-Fosfatidilinositol 4-Quinasa/metabolismo , Aminopiridinas/farmacología , Malaria Falciparum/parasitología , Plasmodium falciparum/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismo , Sulfonas/farmacología , 1-Fosfatidilinositol 4-Quinasa/aislamiento & purificación , Antimaláricos/farmacología , Técnicas de Cultivo de Célula , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Modelos Moleculares , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/aislamiento & purificación , Relación Estructura-Actividad , LevadurasRESUMEN
OBJECTIVE: Hippocampal slice cultures spontaneously develop chronic epilepsy several days after slicing and are used as an in vitro model of post-traumatic epilepsy. Here, we describe a hybrid microfluidic-microelectrode array (µflow-MEA) technology that incorporates a microfluidic perfusion network and electrodes into a miniaturized device for hippocampal slice culture based antiepileptic drug discovery. METHODS: Field potential simulation was conducted to help optimize the electrode design to detect a seizure-like population activity. Epilepsy-on-a-chip model was validated by chronic electrical recording, neuronal survival quantification, and anticonvulsant test. To demonstrate the application of µflow-MEA in drug discovery, we utilized a two-stage screening platform to identify potential targets for antiepileptic drugs. In Stage I, lactate and lactate dehydrogenase biomarker assays were performed to identify potential drug candidates. In Stage II, candidate compounds were retested with µflow-MEA-based chronic electrical assay to provide electrophysiological confirmation of biomarker results. RESULTS AND CONCLUSION: We screened 12 receptor tyrosine kinases inhibitors, and EGFR/ErbB-2 and cFMS inhibitors were identified as novel antiepileptic compounds. SIGNIFICANCE: This epilepsy-on-a-chip system provides the means for rapid dissection of complex signaling pathways in epileptogenesis, paving the way for high-throughput antiepileptic drug discovery.
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Anticonvulsivantes/farmacología , Descubrimiento de Drogas , Electrofisiología/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Análisis de Matrices Tisulares/instrumentación , Encéfalo/citología , Encéfalo/efectos de los fármacos , Descubrimiento de Drogas/instrumentación , Descubrimiento de Drogas/métodos , Epilepsia , HumanosRESUMEN
The Plasmodium falciparum malarial parasite genome appears to encode one and only one phosphatidylinositol 3'-kinase (PI3K), and sequence analysis suggests that the enzyme is a "class III"- or "Vps34"-type PI3K. PfVps34 has generated excitement as a possible druggable target and potentially a key target of artemisinin-based antimalarials. In this study, we optimize the PfVps34 gene for heterologous expression in yeast, purify the protein to homogeneity, use a recently validated quantitative assay for phosphatidylinositol 3'-phosphate production from phosphatidylinositol ( Hassett et al., companion paper; DOI 10.1021/acs.biochem.7b00416 ) to quantify activity and drug inhibition of that activity, and investigate the importance of key residues in the enzyme's catalytic and "N-lobe" domains. Data suggest that PfVps34 is indeed inhibited by artemisinin and related drugs but only under conditions that cleave the drugs' endoperoxide bridge to generate reactive alkylating agents.
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Fosfatidilinositol 3-Quinasas Clase III , Clonación Molecular , Expresión Génica , Plasmodium falciparum , Proteínas Protozoarias , Fosfatidilinositol 3-Quinasas Clase III/biosíntesis , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/genética , Fosfatidilinositol 3-Quinasas Clase III/aislamiento & purificación , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Dominios Proteicos , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Most investigations of phosphatidylinositol 3'-kinase (PI3K) drug inhibition have been via assays based on ADP appearance or ATP consumption (e.g., Liu, Q., et al. ( 2011 ) J. Med. Chem. 54 , 1473 - 1480 ). However, at least some PI3K isoforms show basal ATPase activity in the absence of PI lipid substrate(s), which may complicate quantification of drug potency, isoform specificity of some drugs, and synergy for drug combinations. In this study, we probe the class I vs class III isoform specificity of a selected set of PI3K inhibitors using a simple, inexpensive, semi high-throughput assay that quantifies production of phosphatidylinositol 3'-phosphate (PI3P) from phosphatidylinositol. Results are compared to previous data largely generated using ATPase activity assays. Good agreement between EC50 values computed via ATPase assays vs the reported PI3P formation assay is found for most drugs, but with a few exceptions. Furthermore, for the first time, drug inhibition of class I vs class III enzymes is compared side-by-side with the same assay for the important class I-specific inhibitors GSK2126458 ("Omipalisib") and NVP-BGT226 ("BGT226") currently in clinical development for advanced solid tumors.