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

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

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

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

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

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

Comparison of universal array profile of viral RNA/DNA tested for the corresponding zip-codes.

<p>Normalized average signal intensity for the zip-codes assigned to each virus are presented. The color bars are the signals obtained with the indicated virus (positives). The black bars are the signals produced by the other ten viruses. A signal was considered positive if the intensity of the zip-code spot was at least 10-fold higher than the uniform background level of fluorescence of the array slide. Although a few other viruses produced low-level positive signals for zip18, this did not result in any false positive results since positive signals from at least two addresses was required for a positive identification. In the future, this issue would be rectified by switching to a different zip-code. The average signal intensity for the positives ranged from 31.2 to 123.4, depending on the virus. The average signal intensity for the negatives ranged from 0.3 to 6.2, thus they were not considered positive signals.</p

Schematic of the PCR/LDR assay for detection of VHF viruses.

<p>For each virus (ebolavirus is shown as a representative virus), 1–2 different regions are amplified by RT-PCR using forward and reverse primers, each with minimal degeneracy and all containing universal tails to prevent the formation of primer dimers. Cy-3 labeled downstream LDR primers and single base-discriminating upstream primers with unique zip-code complements (20-30-mers) are targeted to specific sequences/SNPs within the PCR amplicons. Ligation of two adjacent oligonucleotides annealed to a complementary DNA target occurs in the presence of thermostable ligase only if the nucleotides are perfectly matched at the junction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref054" target="_blank">54</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref055" target="_blank">55</a>]. The zip-code complements on the 5’ end of fluorescently labeled LDR products anneal to specific complementary zip-code addresses on a universal array [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref056" target="_blank">56</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138484#pone.0138484.ref057" target="_blank">57</a>]. A positive signal on the universal array is detected as a fluorescent spot. Primers for the ligation reaction were designed targeting 2 or 3 areas within each PCR amplicon. Each virus could produce a maximum of six ligation products, except for VAR and VACC, for which there were a maximum of 5 each. The detection of 2 or more ligation products was required for the detection and identification of a virus. Representative arrays that detect and identify <i>Ebola Zaire</i>, Lassa and Yellow fever viruses are shown.</p

Details of viral cultures and nucleic acids used in the study: RNA viruses.

<p>* The exact sequence of the stock used is not known. The GenBank sequence of the parent strain is indicated.</p><p>Details of viral cultures and nucleic acids used in the study: RNA viruses.</p