Development of Potent Mcl-1 Inhibitors: Structural Investigations on Macrocycles Originating from a DNA-Encoded Chemical Library Screen.

Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein-protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitro potency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59 with a balanced profile, which are suitable for future development toward therapeutic use.

Solid-Phase Synthesis of Caged Luminescent Peptides via Side Chain Anchoring.

The synthesis of caged luminescent peptide substrates remains challenging, especially when libraries of the substrates are required. Most currently available synthetic methods rely on a solution-phase approach, which is less suited for parallel synthesis purposes. We herein present a solid-phase peptide synthesis (SPPS) method for the synthesis of caged aminoluciferin peptides via side chain anchoring of the P1 residue. After the synthesis of a preliminary test library consisting of 40 compounds, the synthetic method was validated and optimized for up to >100 g of resin. Subsequently, two separate larger peptide libraries were synthesized either having a P1 = lysine or arginine residue containing in total 719 novel peptide substrates. The use of a more stable caged nitrile precursor instead of caged aminoluciferin rendered our parallel synthetic approach completely suitable for SPPS and serine protease profiling was demonstrated using late-stage aminoluciferin generation.

Enzymatic glycosylation of triazole-linked GlcNAc/Glc-peptides: synthesis, stability and anti-HIV activity of triazole-linked HIV-1 gp41 glycopeptide C34 analogues.

Long-lasting sweet proteins: The chemoenzymatic synthesis of a triazole (T)-linked glycosylated C34 fragment from HIV-1 gp41 is described. The glycopeptide shows high solubility, excellent fusion inhibition, and as shown in the graph, promising protease resistance. Endoglycosidase-catalyzed transglycosylation of triazole-linked glucose (Glc) and N-acetylglucosamine (GlcNAc)-containing dipeptides and polypeptides was achieved by using synthetic sugar oxazoline as the donor substrate. It was found that both N- and C-linked Glc/GlcNAc-containing triazole derivatives were effective substrates for endo-beta-N-acetylglucosaminidase from Arthrobacter (Endo-A) for transglycosylation; this demonstrates a broad acceptor substrate specificity for Endo-A. This chemoenzymatic method was successfully used for the synthesis of a novel triazole-linked C34 glycopeptide derived from the HIV-1 envelope glycoprotein, gp41. We found that the synthetic C34 glycopeptide possesses potent anti-HIV activity with an IC(50) of 21 nM. The triazole-linked C34 glycopeptide demonstrated a much enhanced stability against protease- and glycoamidase-catalyzed digestion; this shows the protective effects of glycosylation and the stability of the triazole linkage. These favorable properties suggest that the triazole-linked C34 glycopeptide might be valuable for further development as an anti-HIV drug candidate.

Preparation and evaluation of glycosylated arginine-glycine-aspartate (RGD) derivatives for integrin targeting.

Arginine-glycine-aspartate (RGD) derivatives were prepared by a combination of solid-phase and solution-phase synthesis for selective targeting of alpha vbeta 3 integrin expressed in tumors. In order to evaluate the value of a triazole moiety as a proposed amide isostere, the side chain glycosylated cyclic RGD ( cRGD) peptides were synthesized with either a natural amide linkage or a triazole. Affinity of the cRGD constructs for the alpha vbeta 3 integrin was determined in a solid-phase competitive binding assay, showing strong similarity in binding affinity for each of the compounds under evaluation. Furthermore, the in vivo tumor targeting potential of glycosylated cRGD peptides, linked via amide or triazole, was investigated by determining the biodistribution of (125)I-labeled derivatives in mice with tumors expressing alpha vbeta 3. All of the cyclic RGD derivatives showed preferential uptake in the subcutaneous tumors, with the highest tumor-to-blood ratio measured for the triazole-linked glycosylated derivative. The results of the present study are a clear indication of the value of the triazole moiety as a suitable amide isostere in the development of glycosylated peptides as pharmaceuticals.