Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors.

Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay.

Prolonged podocyte depletion in larval zebrafish resembles mammalian focal and segmental glomerulosclerosis.

Focal and segmental glomerulosclerosis (FSGS) is a histological pattern frequently found in patients with nephrotic syndrome that often progress to end-stage kidney disease. The initial step in development of this histologically defined entity is injury and ultimately depletion of podocytes, highly arborized interdigitating cells on the glomerular capillaries with important function for the glomerular filtration barrier. Since there are still no causal therapeutic options, animal models are needed to develop new treatment strategies. Here, we present an FSGS-like model in zebrafish larvae, an eligible vertebrate model for kidney research. In a transgenic zebrafish strain, podocytes were depleted, and the glomerular response was investigated by histological and morphometrical analysis combined with immunofluorescence staining and ultrastructural analysis by transmission electron microscopy. By intravenous injection of fluorescent high-molecular weight dextran, we confirmed leakage of the size selective filtration barrier. Additionally, we observed severe podocyte foot process effacement of remaining podocytes, activation of proximal tubule-like parietal epithelial cells identified by ultrastructural cytomorphology, and expression of proximal tubule markers. These activated cells deposited extracellular matrix on the glomerular tuft which are all hallmarks of FSGS. Our findings indicate that glomerular response to podocyte depletion in larval zebrafish resembles human FSGS in several important characteristics. Therefore, this model will help to investigate the disease development and the effects of potential drugs in a living organism.

Identification of Methionine Aminopeptidase-2 (MetAP-2) Inhibitor M8891: A Clinical Compound for the Treatment of Cancer.

The recently disclosed next generation of reversible, selective, and potent MetAP-2 inhibitors introduced a cyclic tartronic diamide scaffold. However, the lead compound 1a suffered from enterohepatic circulation, preventing further development. Nevertheless, 1a served as a starting point for further optimization. Maintaining potent antiproliferation activity, while improving other compound properties, enabled the generation of an attractive array of new MetAP-2 inhibitors. The most promising derivatives were identified by a multiparameter analysis of the compound properties. Essential for the efficient selection of candidates with in vivo activity was the identification of molecules with a long residence time on the target protein, high permeability, and low efflux ratio not only in Caco-2 but also in the MDR-MDCK cell line. Orally bioavailable, potent, and reversible MetAP-2 inhibitors impede the growth of primary endothelial cells and demonstrated antitumoral activity in mouse models. This assessment led to the nomination of the clinical development compound M8891, which is currently in phase I clinical testing in oncology patients.

XPO1 target occupancy measurements confirm the selinexor recommended phase 2 dose.

XPO1 (exportin 1) is the main nuclear export protein with over 200 different protein cargos. XPO1 is overexpressed in tumor cells and high levels are correlated with poor prognosis. Selective Inhibitor of Nuclear Export (SINE) compounds block nuclear export by inhibiting XPO1. The first SINE compound, selinexor, shows promising anti-cancer activity across hematological and solid tumors in Phase 2 and 3 clinical trials. The 2nd generation SINE compound KPT-8602 is being evaluated as an anti-cancer agent in a Phase 1 clinical trial. To predict patient response to treatment and confirm the selinexor recommended phase 2 dose (RP2D), an assay based on fluorescence cross correlation spectroscopy that measures XPO1 occupancy in cancer cells was developed. Studies comparing cytotoxicity and XPO1 occupancy in cell lines treated with selinexor or KPT-8602 indicated that XPO1 occupancy by both compounds could reach saturation regardless of drug sensitivity. However, higher levels of XPO1 protein correlated with lower sensitivity to SINE compound cytotoxicity. In vivo mouse studies showed XPO1 occupancy could be measured in tumors and was dose-dependent, with >90% target saturation at 10 mg/kg (∼50 mg flat dose in humans). Drug-target occupancy was measured in a dose-response time course and full occupancy occurred by 6 hours at all doses. The duration of occupancy was dose-dependent, where 10-15 mg/kg in mice (∼ 50-75 mg human flat dose) was necessary to maintain XPO1 occupancy up to 48 hours post-dose. These findings confirm the selinexor RP2D of 60 mg for achieving target occupancy and inhibition up to 48 hours.

Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.

Structural and functional insights into the fly microRNA biogenesis factor Loquacious.

In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Loqs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing.

Simultaneous detection of intracellular target and off-target binding of small molecule cancer drugs at nanomolar concentrations.

BACKGROUND AND PURPOSE: In vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off-target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off-target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) as a solution to this problem. EXPERIMENTAL APPROACH: The well-established dihydrofolate reductase inhibitor methotrexate and the kinase inhibitors PD173956 and purvalanol B were conjugated via polyethylene glycol linkers with the fluorophore Cy5. The cellular uptake and subcellular distribution of these compounds in single human cancer-derived cells were investigated by confocal laser scanning microscopy. In addition, molecular interactions inside the cell with the respective target proteins and off-target binding were detected simultaneously in the nanomolar range by FCCS and FCS, respectively, using cells expressing green fluorescent protein fusion proteins of dihydrofolate reductase and Abelson kinase 1. KEY RESULTS: Large differences in the interaction patterns were found for these compounds. For methotrexate-Cy5, drug-target interactions could be detected and dissociation constants determined. In contrast, PD173956-Cy5 showed strong interactions with intracellular high-molecular weight structures, other than its target. CONCLUSIONS AND IMPLICATIONS: The combination of FCS and FCCS provides a powerful means to assess subcellular pharmacokinetics and dynamics of drug candidates at nanomolar concentrations.

The in vitro biological activity of the HLA-DR-binding clinical IgG4 antibody 1D09C3 is a consequence of the disruption of cell aggregates and can be abrogated by Fab arm exchange.

Antibodies of the IgG4 subclass, directed against cell surface antigens have received attention as therapeutic molecules due to their poor induction of the complement system. The MHC class II-directed IgG4 antibody 1D09C3 has been explored for the treatment of lymphomas. The mechanism-of-action is still controversial. Apoptosis induction following HLA-DR engagement has been proposed. However, the validity of these results has been questioned by the observation that antibodies may induce formation of cell aggregates and cell death is induced upon dispersion of these aggregates prior to the quantification of cell death by flow cytometry. Here we address the capacity of 1D09C3 to induce apoptosis in vitro, also taking account of the recently reported Fab arm exchange of IgG4 antibodies. 1D09C3 induces formation of tight cellular aggregates that can only be dispersed at the expense of massive cell damage and death. Using dual color fluorescence cross-correlation spectroscopy (FCCS) we demonstrate that also this antibody undergoes Fab arm exchange in the presence of IgG4. FCCS is a powerful technique to investigate the molecular mechanism of Fab arm exchange using minute amounts of reagents. Following exchange, the functionally monovalent 1D09C3 chimeras loose their ability to induce aggregate formation of HLA-DR-positive cells. Neither functionally monovalent nor bivalent 1D09C3 antibodies induce cell death or apoptosis in myeloma target cells, when microscopy instead of flow cytometry is employed as the analytical technique. Our results indicate that the activity of 1D09C3 in vitro may have been a consequence of assay design rather than an ability to induce HLA-DR-dependent cell death.