Target-Directed Self-Assembly of Homodimeric Drugs Against β‑Tryptase

Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Human tryptase is a donut-shaped tetramer with the active sites facing inward forming a central pore. Bivalent ligands spanning two active sites potently inhibit this configuration, but these large compounds have poor drug-like properties. To overcome some of these challenges, we developed self-assembling molecules, called coferons, which deliver a larger compound in two parts. Using a pharmacophoric core and reversibly binding linkers to span two active sites, we have successfully produced three novel homodimeric tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements in IC₅₀ (0.19 ± 0.08 μM) over controls (5.50 ± 0.09 μM), and demonstrate good activity in mast cell lines. These studies provide validation for this innovative technology that is especially well-suited for the delivery of dimeric drugs to modulate intracellular macromolecular targets

Synthetic Calanolides with Bactericidal Activity against Replicating and Nonreplicating Mycobacterium tuberculosis

It is urgent to introduce new drugs for tuberculosis to shorten the prolonged course of treatment and control drug-resistant Mycobacterium tuberculosis (Mtb). One strategy toward this goal is to develop antibiotics that eradicate both replicating (R) and nonreplicating (NR) Mtb. Naturally occurring (+)-calanolide A was active against R-Mtb. The present report details the design, synthesis, antimycobacterial activities, and structure–activity relationships of synthetic calanolides. We identified potent dual-active nitro-containing calanolides with minimal in vitro toxicity that were cidal to axenic Mtb and Mtb in human macrophages, while sparing Gram-positive and -negative bacteria and yeast. Two of the nitrobenzofuran-containing lead compounds were found to be genotoxic to mammalian cells. Although genotoxicity precluded clinical progression, the profound, selective mycobactericidal activity of these calanolides will be useful in identifying pathways for killing both R- and NR-Mtb, as well as in further structure-based design of more effective and drug-like antimycobacterial agents

The dimeric inhibitors directly bind to Myc and block its interaction with Max.

<p>A) Inhibitors show saturating binding of Myc in SPR experiments. Equilibrium Response Units (RU), normalized to maximal saturated values in individual experiments, are plotted (mean ± SEM) as a function of inhibitor concentration. B) Dose response curves for the inhibition of Myc:Max interaction as determined by ELISA. The data are represented as a fraction of activity compared to a DMSO treated control sample and are plotted as a mean of 2–5 experiments ± SD. The X-axis refers to the concentration of each monomer used.</p

Inhibition of cell-free MYC-MAX heterodimer formation and direct MYC binding^*.

<p>*Average IC₅₀ values (μM) with standard deviation from the MYC-MAX ELISA and average K_D values (μM) from the MYC SPR assay, as described in Experimental Procedures. IC₅₀s and K_Ds of E, N, and C monomers alone are listed first, followed by IC₅₀s and K_Ds from equimolar titrations of combinations of monomers. C11 and C12 are non-dimerizable control compounds corresponding to N11 and N12, respectively.</p><p>Inhibition of cell-free MYC-MAX heterodimer formation and direct MYC binding^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121793#t001fn001" target="_blank">*</a>}.</p

Select combinations of monomers have synergistic activity in a cell proliferation assay.

<p>(A and B) Dose-response curves for two different combinations, E07+N12 (A) and E08+N11 (B)tested in the cell proliferation screen. In each case the dose-response curve for each individual monomer is plotted. The dose-response curves for the predicted additive response (Bliss) and the combination experimental data are plotted with an increasing concentration of E07 orE08 in the presence of N11 or N12 (30 μM). The data is plotted as a mean ± SEM from 3 independent experiments.</p

Overview of the basis for generating self-assembling dimeric inhibitors of the Myc transcription factor.

<p>A) Schematic representation of the self-assembling dimer approach. Individual monomers (Blue and Green) composed of ligand, connector and a paired bioorthoganol linker are delivered to the cells, cross the plasma membrane and react to form an active dimeric inhibitor in the cells. Dimer assembly may occur in the cellular milieu or on the target of interest. B) Schematic representation of the boronic acid/diol equilibria utilized during formation of dimer. Trigonal planar, neutral species are in equilibrium with the charged chiral tetrahedral species. For a given diol, in the cellular milieu at pH 7.4 the equilibria are determined by the pKas of the boronic acids employed and by the pKas of the boronate esters formed. Racemization of the chiral charged species occurs very rapidly and the biological target will select for the most preferred dimer. C) Summary of library design: Structures of the two parent molecules C01 (left) and C02 (right) and attachment positions; connectors are either alkyl chains or PEG-units; R and R’ are linked to the connectors via amide or carbon bonds; synthetic details of selected library members are provided in the supplementary experimental procedures.</p

Dimeric inhibitors of Myc drive anti-proliferative effects in Myc over-expressing cell lines that are correlated with a decrease in Myc protein levels.

<p>A) Daudi cells were treated with the indicated compounds or combinations for 72 hours and cell viability measured (left panel, * p< 0.05, ** p<0.001, <i>ns</i> not significant). In a parallel experiment Daudi cells were treated with E08+N11 or E08+C11 combinations for the indicated times and protein lysates probed with the indicated antibodies (right panel). E08 was used at 10μM and N11 or C11 were used at 30μM. (B) Raji and (C) K562 cells were treated and analyzed as detailed in (A).</p

The dimeric inhibitors block Myc:Max but not Max:Max binding to DNA.

<p>Gel mobility shift assay showing the effects on Myc:Max DNA complex formation by the dimeric inhibitor E07+N12 (A) and the non-dimerizable control combination E07+C12 (B). The bands that represent protein-DNA complex or naked DNA are shown on the right hand side of each panel. The concentrations indicated are in μM.</p