Emilio Margolles-Clark

The food colorant erythrosine is a promiscuous protein―protein interaction inhibitor

Following our observation that erythrosine B (FD&C Red No. 3) is a relatively potent inhibitor of the TNF-R-TNFα and CD40-CD154 protein-protein interactions, we investigated whether this inhibitory activity extends to any other protein-protein interactions (PPI) as well as whether any other approved food colors possess such inhibitory activity. We found erythrosine, a poly-iodinated xanthene dye, to be a non-specific promiscuous inhibitor of a number of PPIs within the tumor necrosis factor superfamily (TNF-R-TNFα, CD40-CD154, BAFF-R-BAFF, RANK-RANKL, OX40-OX40L, 4-1BB-4-1BBL) as well as outside of it (EGF-R-EGF) with a remarkably consistent median inhibitory concentration (IC(50)) in the 2-20 μM (approximately 2-20mg/L) range. In agreement with this, erythrosine also showed cellular effects including clear cytotoxic effects around this concentration range (IC₅₀≈50 μM). Among the seven FDA-approved food colorants, only erythrosine showed consistent PPI inhibitory activity in the sub-100 μM range, which might also explain (at least partially) why it also has the lowest approved acceptable daily intake (ADI) (0.1 mg/kg body weight/day). Among a number of xanthene structural analogs of erythrosine tested for activity, rose Bengal, a food colorant approved in Japan, showed similar, maybe even more pronounced, promiscuous inhibitory activity, whereas fluorescein was inactive and gallein, phloxine, and eosin were somewhat active in some of the assays.

Role of four calcium transport proteins, encoded by nca-1, nca-2, nca-3, and cax, in maintaining intracellular calcium levels in Neurospora crassa.

ABSTRACT We have examined the distribution of calcium in Neurospora crassa and investigated the role of four predicted calcium transport proteins. The results of cell fractionation experiments showed 4% of cellular calcium in mitochondria, approximately 11% in a dense vacuolar fraction, 40% in an insoluble form that copurifies with microsomes, and 40% in a high-speed supernatant, presumably from large vacuoles that had broken. Strains lacking NCA-1, a SERCA-type Ca2+-ATPase, or NCA-3, a PMC-type Ca2+-ATPase, had no obvious defects in growth or distribution of calcium. A strain lacking NCA-2, which is also a PMC-type Ca2+-ATPase, grew slowly in normal medium and was unable to grow in high concentrations of calcium tolerated by the wild type. Furthermore, when grown in normal concentrations of calcium (0.68 mM), this strain accumulated 4- to 10-fold more calcium than other strains, elevated in all cell fractions. The data suggest that NCA-2 functions in the plasma membrane to pump calcium out of the cell. In this way, it resembles the PMC-type enzymes of animal cells, not the Pmc1p enzyme in Saccharomyces cerevisiae that resides in the vacuole. Strains lacking the cax gene, which encodes a Ca2+/H+ exchange protein in vacuolar membranes, accumulate very little calcium in the dense vacuolar fraction but have normal levels of calcium in other fractions. The cax knockout strain has no other observable phenotypes. These data suggest that “the vacuole” is heterogeneous and that the dense vacuolar fraction contains an organelle that is dependent upon the CAX transporter for accumulation of calcium, while other components of the vacuolar system have multiple calcium transporters.

Small-molecule costimulatory blockade: organic dye inhibitors of the CD40–CD154 interaction

Costimulatory blockade is one of the most promising therapeutic targets in autoimmune diseases as well as in transplant recipients, and inhibition of the cluster of differentiation (CD)40–CD154 interaction, which is required for T cell activation and development of an effective immune response, is particularly promising in islet transplant recipients. Here, we report the ability of several small-molecule organic dyes to concentration dependently inhibit this interaction with IC50 values in the low-micromolar range. They were found to be considerably more active in inhibiting this interaction than the tumor necrosis factor (TNF)-R1-TNF-α or B cell-activating factor (BAFF)-R-BAFF interaction, which are members of the same family. They specifically inhibited CD154-induced cell responses in human B cells as well as in THP-1 myeloid cells, which can serve as surrogate dendritic cells, at concentrations well below their cytotoxic concentrations determined in the same cells. Flow cytometry experiments confirmed their ability to inhibit the CD154-induced, but not the Staphylococcus aureus Cowan I- or phorbol 12-myristate 13-acetate-induced increase in the surface expression of CD54, CD40, and major histocompatibility complex class II. Accordingly, these compounds can be useful not only for experimental investigations involving the inhibition of the CD40–CD154 costimulatory interaction but can also provide important structure–activity relationship information and can serve as the starting point of a targeted drug discovery program.

Small‐molecule modulators of the OX40–OX40 ligand co‐stimulatory protein–protein interaction

The OX40–OX40L protein–protein interaction (PPI) is an important cell‐surface signalling co‐stimulatory regulator within the TNFR superfamily (TNFRSF) and a promising therapeutic target for immunomodulation. PPIs are difficult to modulate using small‐molecules. Here, we describe the identification of a small‐molecule OX40 modulator and confirm its partial agonist character.

Effective and specific inhibition of the CD40-CD154 costimulatory interaction by a naphthalenesulphonic acid derivative.

Costimulatory interactions are important regulators of T‐cell activation and, hence, promising therapeutic targets in autoimmune diseases as well as in transplant recipients. Following our recent identification of the first small‐molecule inhibitors of the CD40–CD154 costimulatory protein–protein interaction (J Mol Med 87, 2009, 1133), we continued our search within the chemical space of organic dyes, and we now report the identification of the naphthalenesulphonic acid derivative mordant brown 1 as a more active, more effective, and more specific inhibitor. Flow cytometry experiments confirmed its ability to concentration‐dependently inhibit the CD154(CD40L)‐induced cellular responses in human THP‐1 cells at concentrations well below cytotoxic levels. Binding experiments showed that it not only inhibits the CD40–CD154 interaction with sub‐micromolar activity, but it also has considerably more than 100‐fold selectivity toward this interaction even when compared to other members of the tumor necrosis factor superfamily pairs such as TNF‐R1–TNF‐α, BAFF‐R(CD268)–BAFF(CD257/BLys), OX40(CD134)–OX40L(CD252), RANK(CD265)–RANKL(CD254/TRANCE), or 4‐1BB(CD137)–4‐1BBL. There is now sufficient structure‐activity relationship information to serve as the basis of a drug discovery initiative targeting this important costimulatory interaction.

Organic dyes as small molecule protein-protein interaction inhibitors for the CD40-CD154 costimulatory interaction.

It is becoming increasingly clear that small molecules can often act as effective protein–protein interaction (PPI) inhibitors, an area of increasing interest for its many possible therapeutic applications. We have identified several organic dyes and related small molecules that (i) concentration‐dependently inhibit the important CD40–CD154 costimulatory interaction with activities in the low micromolar (µM) range, (ii) show selectivity toward this particular PPI, (iii) seem to bind on the surface of CD154, and (iv) concentration‐dependently inhibit the CD154‐induced B cell proliferation. They were identified through an iterative activity screening/structural similarity search procedure starting with suramin as lead, and the best smaller compounds, the main focus of the present work, achieved an almost 3‐fold increase in ligand efficiency (ΔG0/nonhydrogen atom = 0.8 kJ/NnHa) approaching the average of known promising small‐molecule PPI inhibitors (∼1.0 kJ/NnHa). Since CD154 is a member of the tumor necrosis factor (TNF) superfamily of cell surface interaction molecules, inhibitory activities on the TNF‐R1–TNF‐α interactions were also determined to test for specificity, and the compounds selected here all showed more than 30‐fold selectivity toward the CD40–CD154 interaction. Because of their easy availability in various structural scaffolds and because of their good protein‐binding ability, often explored for tissue‐specific staining and other purposes, such organic dyes can provide a valuable addition to the chemical space searched to identify small molecule PPI inhibitors in general. Copyright

Research Article: Effective and Specific Inhibition of the CD40–CD154 Costimulatory Interaction by a Naphthalenesulphonic Acid Derivative

Costimulatory interactions are important regulators of T‐cell activation and, hence, promising therapeutic targets in autoimmune diseases as well as in transplant recipients. Following our recent identification of the first small‐molecule inhibitors of the CD40–CD154 costimulatory protein–protein interaction (J Mol Med 87, 2009, 1133), we continued our search within the chemical space of organic dyes, and we now report the identification of the naphthalenesulphonic acid derivative mordant brown 1 as a more active, more effective, and more specific inhibitor. Flow cytometry experiments confirmed its ability to concentration‐dependently inhibit the CD154(CD40L)‐induced cellular responses in human THP‐1 cells at concentrations well below cytotoxic levels. Binding experiments showed that it not only inhibits the CD40–CD154 interaction with sub‐micromolar activity, but it also has considerably more than 100‐fold selectivity toward this interaction even when compared to other members of the tumor necrosis factor superfamily pairs such as TNF‐R1–TNF‐α, BAFF‐R(CD268)–BAFF(CD257/BLys), OX40(CD134)–OX40L(CD252), RANK(CD265)–RANKL(CD254/TRANCE), or 4‐1BB(CD137)–4‐1BBL. There is now sufficient structure‐activity relationship information to serve as the basis of a drug discovery initiative targeting this important costimulatory interaction.

Small-molecule modulators of the OX40OX40 ligand co-stimulatory proteinprotein interaction

The OX40–OX40L protein–protein interaction (PPI) is an important cell‐surface signalling co‐stimulatory regulator within the TNFR superfamily (TNFRSF) and a promising therapeutic target for immunomodulation. PPIs are difficult to modulate using small‐molecules. Here, we describe the identification of a small‐molecule OX40 modulator and confirm its partial agonist character.

Small-molecule modulators of the OX40-OX40 ligand co-stimulatory protein-protein interaction: Small-molecule OX40-OX40L modulators

The OX40–OX40L protein–protein interaction (PPI) is an important cell‐surface signalling co‐stimulatory regulator within the TNFR superfamily (TNFRSF) and a promising therapeutic target for immunomodulation. PPIs are difficult to modulate using small‐molecules. Here, we describe the identification of a small‐molecule OX40 modulator and confirm its partial agonist character.

Research Article: Effective and Specific Inhibition of the CD40-CD154 Costimulatory Interaction by a Naphthalenesulphonic Acid Derivative: Inhibition of the CD40-CD154 Costimulatory Interaction

Costimulatory interactions are important regulators of T‐cell activation and, hence, promising therapeutic targets in autoimmune diseases as well as in transplant recipients. Following our recent identification of the first small‐molecule inhibitors of the CD40–CD154 costimulatory protein–protein interaction (J Mol Med 87, 2009, 1133), we continued our search within the chemical space of organic dyes, and we now report the identification of the naphthalenesulphonic acid derivative mordant brown 1 as a more active, more effective, and more specific inhibitor. Flow cytometry experiments confirmed its ability to concentration‐dependently inhibit the CD154(CD40L)‐induced cellular responses in human THP‐1 cells at concentrations well below cytotoxic levels. Binding experiments showed that it not only inhibits the CD40–CD154 interaction with sub‐micromolar activity, but it also has considerably more than 100‐fold selectivity toward this interaction even when compared to other members of the tumor necrosis factor superfamily pairs such as TNF‐R1–TNF‐α, BAFF‐R(CD268)–BAFF(CD257/BLys), OX40(CD134)–OX40L(CD252), RANK(CD265)–RANKL(CD254/TRANCE), or 4‐1BB(CD137)–4‐1BBL. There is now sufficient structure‐activity relationship information to serve as the basis of a drug discovery initiative targeting this important costimulatory interaction.