Search

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these 'design rules' to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif., (© 2024. Merck & Co., Inc., Rahway, NJ, USA and its affiliates and Tsz Ying Yuen, Diana Spiegelberg, Pietro G. Aronica, Yee Hwee Lim, Marika Nestor, Chandra S. Verma, David P. Lane, Srinivasaraghavan Kannan, Christopher J. Brown, Charles W. Johannes.)

Purpose: To develop a novel, target agnostic liposome click membrane permeability assay (LCMPA) using liposome encapsulating copper free click reagent dibenzo cyclooctyne biotin (DBCO-Biotin) to conjugate azido modified peptides that may effectively translocate from extravesicular space into the liposome lumen., Method: DBCO-Biotin liposomes were prepared with egg phosphatidylcholine and cholesterol by lipid film rehydration, freeze/thaw followed by extrusion. Size of DBCO-Biotin liposomes were characterized with dynamic light scattering., Results: The permeable peptides representing energy independent mechanism of permeability showed higher biotinylation in LCMPA. Individual peptide permeability results from LCMPA correlated well with shifts in potency in cellular versus biochemical assays (i.e., cellular/ biochemical ratio) demonstrating quantitative correlation to intracellular barrier in intact cells., Conclusion: The study provides a novel membrane permeability assay that has potential to evaluate energy independent transport of diverse peptides.

Macrocyclic α-helical peptides have emerged as a compelling new therapeutic modality to tackle targets confined to the intracellular compartment. Within the scope of hydrocarbon-stapling there has been significant progress to date, including the first stapled α-helical peptide to enter into clinical trials. The principal design concept of stapled α-helical peptides is to mimic a cognate (protein) ligand relative to binding its target via an α-helical interface. However, it was the proclivity of such stapled α-helical peptides to exhibit cell permeability and proteolytic stability that underscored their promise as unique macrocyclic peptide drugs for intracellular targets. This perspective highlights key learnings as well as challenges in basic research with respect to structure-based design, innovative chemistry, cell permeability and proteolytic stability that are essential to fulfill the promise of stapled α-helical peptide drug development., (Copyright © 2018. Published by Elsevier Ltd.)

Traditionally, computing the binding affinities of proteins to even relatively small and rigid ligands by free-energy methods has been challenging due to large computational costs and significant errors. Here, we apply a new molecular simulation acceleration method called MELD (Modeling by Employing Limited Data) to study the binding of stapled α-helical peptides to the MDM2 and MDMX proteins. We employ free-energy-based molecular dynamics simulations (MELD-MD) to identify binding poses and calculate binding affinities. Even though stapled peptides are larger and more complex than most protein ligands, the MELD-MD simulations can identify relevant binding poses and compute relative binding affinities. MELD-MD appears to be a promising method for computing the binding properties of peptide ligands with proteins.

Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

A series of novel nonpeptide inhibitors of the pp60(c-Src) (Src) SH2 domain is described that exploit multifunctional group replacement of the phenylphosphate moiety of phosphotyrosine (pTyr). Relative to an x-ray structure of citrate complexed to the pTyr binding site of the Src SH2 domain, these nonpeptide ligands illustrate the systematic replacement of the phosphate group by multiple nonhydrolyzable, mono- or dianionic functionalities. Specifically, several phenylalanine (Phe) analogs incorporating key 4' and 3' substituents were synthesized and incorporated into a bicyclic benzamide template previously reported (W. C. Shakespeare et al., Proceedings of the National Academy of Science USA, 2000, Vol. 97, pp. 9373-9378). These pTyr mimetics included 4',3'-diphosphono-Phe (Dpp), 4',3'-dicarboxymethyloxy-Phe (Dcp), and 4'-phosphono-3'-carboxymethyloxy-Phe (Cpp). Noteworthy were nonpeptide inhibitors 8-11 that were 5- to 10-fold more potent than the cognate tetrapeptide ligand Ac-pTyr-Glu-Glu-Ile-NH(2) in binding to the Src SH2 domain., (Copyright 2004 Wiley Periodicals, Inc.)

The structural and functional characterization of Src homology-2 (SH2) domains and their relationship to catalytic proteins (e.g., kinases, phosphatases, and lipases) or non-catalytic proteins (e.g., upstream adapters, and downstream transcription factors) has significantly impacted our understanding of signal transduction pathways and the identification of promising therapeutic targets for drug discovery. Such SH2-containing proteins are known to be intimately involved in the regulation of a number of cellular processes, including growth, mitogenesis, motility, metabolism, and gene transcription. Molecular recognition and biochemical selectivity exists for various SH2 domains based on their binding to phosphotyrosine (pTyr) and contiguous C-terminal amino acids of cognate protein 'partners' in a sequence-dependent manner (i.e., -pTyr-AA(1)-AA(2)-AA(3)-) which result in the formation of signal transduction protein complexes in cells. In recent years, drug discovery efforts have advanced peptidomimetic and nonpeptide inhibitors of such protein-protein interactions based on mimicking pTyr-containing peptide ligands as well as SH2 structure-based de novo design of nonpeptide templates that can capture key binding sites on the target protein. Noteworthy are peptidomimetic and nonpeptide inhibitors of Src, Lck, Grb2, PI-3K, and Zap70 from pioneering efforts that led to the first examples of cellularly and in vivo active SH2 inhibitors. This mini-review highlights key achievements in SH2 inhibitor drug discovery with an emphasis on peptidomimetic and nonpeptide lead compounds in terms of structure-based design, key chemical and biological properties, and proof-of-concept studies relative to further defining the role(s) of SH2 domains in signal transduction processes, cellular functions, and in vivo disease models.

Three-dimensional molecular models of the human melanocortin receptor (hMC1R) have been developed based upon the electron cryo-microscopic structure of bacteriorhodopsin and the electron density footprint of bovine rhodopsin. alpha-Melanocyte-stimulating hormone, Ac-Ser-Tyr-Ser-Met4-Glu-His-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2 (alpha-MSH, alpha-melanotropin), and the superpotent, prolonged acting agonists, Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2 (NDP-MSH) and Ac-Nle4-c[Asp5-His6-DPhe7-Arg8-Trp9-Lys10]-NH2 (MTII), have been modeled into the proposed binding sites with specific ligand-receptor interactions identified. The melanotropin sidechain pharmacophores, DPhe7 and Trp9, are proposed to interact with a hydrophobic network of receptor aromatic residues in transmembrane regions 4, 5, 6, and 7. In addition, a hydrophilic network involving the ligand Arg8 and polar receptor residues located in transmembrane regions 2 and 3 were identified. Biological studies on alpha-MSH, NDP-MSH, MTII, and related peptides have been correlated with the proposed hMC1R model in terms of agonism, affinity, and prolongation. Finally, limited MC1R mutagenesis studies comparing alpha-MSH and NDP-MSH are interpreted within the context of the proposed hMC1R models.

The independent subsite model is widely used for the design of peptide inhibitors of enzymes with extended active sites. This model assumes that the subsites are independent of each other and that the free energies of binding contributed by the several subsites are additive. We questioned the strict application of this model for structure-activity studies, since one can, a priori, conceive of likely deviations from this model. Accordingly, we tested the independent subsite model by measuring the thermodynamic binding parameters of a series of peptide inhibitors of human renin. This enzyme-inhibitor system was chosen as a model by virtue of the high degree of specificity of renin for its natural substrate, angiotensinogen, and the availability of a large number of structurally similar peptide inhibitors. Although we found the general mode of binding of these renin inhibitors to be primarily hydrophobic, serious deviations from additivity and independent subsite model constraints were observed. We conclude that an important determinant of binding is most probably the conformation assumed by the peptide inhibitor in solution. Thus, we suggest that caution be exercised in using affinity constants to assess the interactions of peptide inhibitors with human renin and possibly with other enzymes having extended binding sites. Furthermore, the thermodynamic parameters of a class of compounds provide more information as to the mode of binding of ligands to their respective receptors than do dissociation constants.

The structure-conformation relationships of a series of angiotensinogen6-13 (ANG6-13, His-Pro-Phe-His-Leu-Val-Ile-His) congeners substituted by Nin-For-Trp (Ftr), D-Ftr or Trp at the N-terminus, Tyr at the C-terminus and Phe psi[CH2NH]Phe at the P1-P'1 cleavage site (i.e. Leu10-Val11) were studied using resonance energy transfer coupled with molecular modeling of the peptide conformation using macromolecular energy refinement and dynamics simulation. Average end-to-end intramolecular distances (r) of the peptides in solution were determined by fluorescence spectroscopy. For example, Ac-Ftr-Pro-Phe-His-Phe psi[CH2NH]Phe-Val-Tyr-NH2 (U-70714E) gave an average intramolecular donor (Tyr)-acceptor (Ftr) distance of 16.3A in aqueous solution. This experimental value was consistent with a distance of 17.9 A determined by molecular modeling of U-70714E to a human renin 3-D structure (developed from known homologous aspartyl protease inhibitor X-ray crystallographic data) followed by simulation of the solution phase conformation of the peptide. An extended backbone secondary structure of U-70714E is suggested from these studies and the relationship(s) of structure-conformation to structure-activity was explored by analysis of several congeners of U-70714E, a potent (IC50 = 3.0 X 10(-9)M) inhibitor of human renin in vitro.