In-solution enrichment identifies peptide inhibitors of protein-protein interactions.

The use of competitive inhibitors to disrupt protein-protein interactions (PPIs) holds great promise for the treatment of disease. However, the discovery of high-affinity inhibitors can be a challenge. Here we report a platform for improving the affinity of peptide-based PPI inhibitors using non-canonical amino acids. The platform utilizes size exclusion-based enrichment from pools of synthetic peptides (1.5-4 kDa) and liquid chromatography-tandem mass spectrometry-based peptide sequencing to identify high-affinity binders to protein targets, without the need for 'reporter' or 'encoding' tags. Using this approach-which is inherently selective for high-affinity binders-we realized gains in affinity of up to ~100- or ~30-fold for binders to the oncogenic ubiquitin ligase MDM2 or HIV capsid protein C-terminal domain, which inhibit MDM2-p53 interaction or HIV capsid protein C-terminal domain dimerization, respectively. Subsequent macrocyclization of select MDM2 inhibitors rendered them cell permeable and cytotoxic toward cancer cells, demonstrating the utility of the identified compounds as functional PPI inhibitors.

Author Correction: In-solution enrichment identifies peptide inhibitors of protein-protein interactions.

In the version of this article originally published, the peptide sequences of compounds 90, 92 and 93 in Fig. 5b and Supplementary Table 7 contained several errors. In Fig. 5b, position 6 of compound 90 should be Tyr instead of Phe. In both Fig. 5b and Supplementary Table 7, position 9 of compounds 92 and 93 should be Gln instead of Glu. Additionally, the surname of co-author Anupam Bandyopadhyay was incorrectly spelled as Bandyopdhyay. The errors have been corrected in the HTML and PDF versions of the paper and in the Supplementary Information PDF.

Supramolecular Ladder Assemblies as a Model for Probing Electronic Interactions between Multiple Stacked π-Conjugated Systems.

A series of mono-, di-, and tri-topic receptors in which H-bonding sites, complementary to those of barbituric acid (BA), are fused is used to induce the supramolecular assembly of n×m ladders containing 1, 2, or 3 triphenylenevinylene units appended with BA. The topological constraint enforced by the architectures induces through-space interactions between the electroactive moieties that are reflected in the electronic absorption and emission spectrum. The n=2, m=2 or m=3 architectures undergo two single electron oxidation events, indicative of the formation of the corresponding mono- and di-radical cation species with comproportionation constants of 340 and 70, respectively. Comparison of the electrochemical potentials suggests that the charges are delocalized over the electroactive units in the assembly.

Antibody-Bactericidal Macrocyclic Peptide Conjugates To Target Gram-Negative Bacteria.

To combat antimicrobial infections, new active molecules are needed. Antimicrobial peptides, ever abundant in nature, are a fertile starting point to develop new antimicrobial agents but suffer from low stability, low specificity, and off-target toxicity. These drawbacks have limited their development. To overcome some of these limitations, we developed antibody-bactericidal macrocyclic peptide conjugates (ABCs), in which the antibody directs the bioactive macrocyclic peptide to the targeted Gram-negative bacteria. We used cysteine SN Ar chemistry to synthesize and systematically study a library of large (>30-mer) macrocyclic antimicrobial peptides (mAMPs) to discover variants with extended proteolytic stability in human serum and low hemolytic activity while maintaining bioactivity. We then conjugated, by using sortase A, these bioactive variants onto an Escherichia coli targeted monoclonal antibody. We found that these ABCs had minimized hemolytic activity and were able to kill E. coli at nanomolar concentrations. Our findings suggest macrocyclic peptides if fused to antibodies may facilitate the discovery of new agents to treat bacterial infections.

Palladium-Mediated Arylation of Lysine in Unprotected Peptides.

A mild method for the arylation of lysine in an unprotected peptide is presented. In the presence of a preformed biarylphosphine-supported palladium(II)-aryl complex and a weak base, lysine amino groups underwent C-N bond formation at room temperature. The process generally exhibited high selectivity for lysine over other amino acids containing nucleophilic side chains and was applicable to the conjugation of a variety of organic compounds, including complex drug molecules, with an array of peptides. Finally, this method was also successfully applied to the formation of cyclic peptides by macrocyclization.

Metal-Coordination-Assisted Folding and Guest Binding in Helical Aromatic Oligoamide Molecular Capsules.

The development of foldamer-based receptors is driven by the design of monomers with specific properties. Herein, we introduce a pyridazine-pyridine-pyridazine diacid monomer and its incorporation into helical aromatic oligoamide foldamer containers. This monomer codes for a wide helix diameter and can sequester metal ions on the inner wall of the helix cavity. Crystallographic studies and NMR titrations show that part of the metal coordination sphere remains available and may then promote the binding of a guest within the cavity. In addition to metal coordination, binding of the guest is assisted by cooperative interactions with the helix host, thereby resulting in significant enhancements depending on the foldamer sequence, and in slow guest capture and release on the NMR time scale. In the absence of metal ions, the pyridazine-pyridine-pyridazine monomer promotes an extended conformation of the foldamer that results in aggregation, including the formation of an intertwined duplex.

Iterative Evolution of an Abiotic Foldamer Sequence for the Recognition of Guest Molecules with Atomic Precision.

A synthetic helical aromatic oligoamide foldamer receptor with high affinity and selectivity for tartaric acid was subjected to a structure-based evolution of its sequence via mutations, additions, and deletions of monomers to produce a new receptor having high affinity and selectivity for malic acid, a guest that differs from tartaric acid by a single oxygen atom. Seven iteratively modified sequences were synthesized. Detailed structural investigations of host-guest complexes were carried out systematically to guide the design of the next generation. A first outcome was a reversal of selectivity of the receptors, with a starting preference for tartaric acid over malic acid of over 10(2) and an ending sequence showing a preference for malic acid over tartaric acid of over 10(2). Another outcome was a very strong enhancement of the affinity for malic acid, despite the fact that it has fewer recognition features for binding through polar interactions such as hydrogen bonds. Such a level of discrimination between resembling guests exemplifies the amenability of foldamers to outstanding achievements in molecular recognition. Altogether, our results demonstrate the viability of a rational receptor design approach that exploits the modularity of foldamer sequences and, in the case of aromatic amide foldamers, their amenability to structural elucidation, their relative ease of synthesis, and the predictability of their structure.

Nitrogen Arylation for Macrocyclization of Unprotected Peptides.

We describe an efficient and mild method for the synthesis of macrocyclic peptides via nitrogen arylation from unprotected precursors. Various electrophiles and lysine-based nucleophiles were investigated and showed high-yielding product formation, even for a macrocyclization scan with 14 variants. We found that nitrogen-linked aryl products were more stable to base and oxidation when compared to thiol arylated species, thereby highlighting the utility of this methodology. Finally, N-aryl macrocyclization was performed on a p53 peptide inhibitor of MDM2 and resulted in identification of a nanomolar binder with improved proteolytic stability and cell permeability.

Polar solvent effects on tartaric acid binding by aromatic oligoamide foldamer capsules.

Aromatic oligoamide sequences able to fold into single helical capsules were functionalized with two types of side chains to make them soluble in various solvents such as chloroform, methanol or water and their propensity to recognize tartaric acid was evaluated. The binding affinities to tartaric acid and binding thermodynamics in different media were investigated by variable temperature (1)H NMR and ITC experiments, the two methods giving consistent results. We show that tartaric acid binding mainly rests on enthalpically favourable polar interactions that were found to be sufficiently strong to be effective in the presence of a polar aprotic solvent (DMSO) and even in pure methanol. Binding in water was very weak. The stronger binding interactions were found to be more susceptible to the effect of competitive solvents and compensated by unfavourable entropic effects. Thus, the best host in a less polar medium eventually was found to be the worst host in protic solvents. An interesting case of entropically driven binding was evidenced in methanol.

Tuning the guest-binding ability of a helically folded capsule by in situ modification of the aromatic oligoamide backbone.

Starting from a previously described aromatic oligoamide helically folded capsule that binds tartaric acid with high affinity and diastereoselectivity, we demonstrate the feasibility of the direct in situ modification of the helix backbone, which results in a conformational change that reduces its affinity for guests by two orders of magnitude. Specifically, ring contraction of the central pyridazine unit into a pyrrole in the full helical sequence was investigated by using electrochemical and chemical processes. The sequence containing the pyrrole was synthesized independently in a convergent manner to ascertain its structure. The conformation of the pyrrolic folded capsule was elucidated in the solid state by X-ray crystallography and in solution by using (1)H and (13)C NMR spectroscopy. Solution studies revealed an unanticipated solvent-dependent equilibrium between the anti-anti and syn-syn conformations of the pyrrole ring with respect to its two adjacent pyridine units. Titrations of the pyrrole-containing sequence monitored by (1)H NMR spectroscopy confirmed the expected drop in affinity for tartaric acid and malic acid that arises from the conformation change in the backbone that follows the replacement of the pyridazine by a pyrrole. The reduction of the pyridazine to a pyrrole was characterized by cyclic voltammetry both on the entire sequence and on a shorter precursor. The lower cathodic potential of the precursor made its preparative-scale electroreduction possible. Direct in situ modification of the pyridazine within the entire capsule sequence was achieved chemically by using zinc in acetic acid.

Structure elucidation of host-guest complexes of tartaric and malic acids by quasi-racemic crystallography.

Racemic on the outside, but not inside: Aromatic-foldamer hosts are enantiomers and as such prefer to co-crystallize even though the guests (e.g. L-tartaric acid, see picture) in each host are not present as enantiomers. This behavior allows a one-step structure elucidation of diastereomeric and quasi-racemic structures in the solid state.

Aromatic foldamers as scaffolds for metal second coordination sphere design.

As metalloproteins exemplify, the chemical and physical properties of metal centers depend not only on their first but also on their second coordination sphere. Installing arrays of functional groups around the first coordination sphere of synthetic metal complexes is thus highly desirable, but it remains a challenging objective. Here we introduce a novel approach to produce tailored second coordination spheres. We used bioinspired artificial architectures based on aromatic oligoamide foldamers to construct a rigid, modular and well-defined environment around a metal complex. Specifically, aza-aromatic monomers having a tethered [2Fe-2S] cluster have been synthesized and incorporated in conical helical foldamer sequences. Exploiting the modularity and predictability of aromatic oligoamide structures allowed for the straightforward design of a conical architecture able to sequester the metal complex in a confined environment. Even though no direct metal complex-foldamer interactions were purposely designed in this first generation model, crystallography, NMR and IR spectroscopy concurred to show that the aromatic oligoamide backbone alters the structure and fluxional processes of the metal cluster.

Iterative design of a helically folded aromatic oligoamide sequence for the selective encapsulation of fructose.

The ab initio design of synthetic molecular receptors for a specific biomolecular guest remains an elusive objective, particularly for targets such as monosaccharides, which have very close structural analogues. Here we report a powerful approach to produce receptors with very high selectivity for specific monosaccharides and, as a demonstration, we develop a foldamer that selectively encapsulates fructose. The approach uses an iterative design process that exploits the modular structure of folded synthetic oligomer sequences in conjunction with molecular modelling and structural characterization to inform subsequent refinements. Starting from a first-principles design taking size, shape and hydrogen-bonding ability into account and using the high predictability of aromatic oligoamide foldamer conformations and their propensity to crystallize, a sequence that binds to ß-D-fructopyranose in organic solvents with atomic-scale complementarity was obtained in just a few iterative modifications. This scheme, which mimics the adaptable construction of biopolymers from a limited number of monomer units, provides a general protocol for the development of selective receptors.