Design and Biochemical Characterization of Peptidic Inhibitors of the Myb/p300 Interaction.

The Myb transcription factor is involved in the proliferation of hematopoietic cells, and deregulation of its expression can lead to cancers such as leukemia. Myb interacts with various proteins, including the histone acetyltransferases p300 and CBP. Myb binds to a small domain of p300, the KIX domain (p300KIX), and inhibiting this interaction is a potential new drug discovery strategy in oncology. The available structures show that Myb binds to a very shallow pocket of the KIX domain, indicating that it might be challenging to identify inhibitors of this interaction. Here, we report the design of Myb-derived peptides which interact with p300KIX. We show that by mutating only two Myb residues that bind in or near a hotspot at the surface of p300KIX, it is possible to obtain single-digit nanomolar peptidic inhibitors of the Myb/p300KIX interaction that bind 400-fold tighter to p300KIX than wildtype Myb. These findings suggest that it might also be possible to design potent low molecular-weight compounds to disrupt the Myb/p300KIX interaction.

Important Considerations Related to Permeability of Peptides.

This review on intracellular delivery and oral bioavailability of peptides reflects a number of principal investigations at Novartis. Our studies were aimed at either understanding features enabling peptides to interfere with intracellular protein-protein interactions, or to achieve a more patient-friendly delivery by the oral route. In the light of these objectives, we have also spent some effort on assay development to come up with alternative methods for monitoring cellular peptide uptake. This summary of our insights is intended to help in the assessment and development of peptide therapeutics requiring membrane transition.

A new perspective on the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo.

Peptides constitute an important class of drugs for the treatment of multiple metabolic, oncological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel "Split Luciferin Peptide" (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a d-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development.

Late-Stage Functionalization of Peptides and Cyclopeptides Using Organozinc Reagents.

We report a new late-stage functionalization of small peptides and cyclopeptides relying on the Negishi cross-coupling of readily prepared iodotyrosine- or iodophenylalanine-containing peptides with aryl-, heteroaryl-, and alkylzinc pivalates or halides. In silico and in vitro determinations of membrane permeability parameters of the modified cyclopeptides showed that in most cases, the solubility was improved by the introduction of polar pyridyl units while the cell-membrane permeability was maintained.

Modulation of Oral Bioavailability and Metabolism for Closely Related Cyclic Hexapeptides.

ABSTRACT: Recently, a variety of studies concerned with the permeability and oral bioavailability of cyclic peptides have been reported. In particular, strategies aiming at modifying peptides to maintain or to enhance solubility while enabling permeability constitute a significant challenge, but are of high interest to ensure a smooth drug discovery process. Current methodologies include N-methylation, matching of hydrogen bonding acceptors and donors across the macrocycle, and additional masking of polarity. In this study, we investigate further the pivotal effects of shielding on permeability and studied the metabolism of the corresponding peptides in more detail by comparing peptide concentrations in the portal versus the jugular vein in rats. Interestingly, minor changes in one particular side chain impacts both permeability and liver metabolism.

Evaluating Cellular Drug Uptake with Fluorescent Sensor Proteins.

We are introducing a new approach to evaluate cellular uptake of drugs and drug candidates into living cells. The approach is based on converting the protein target of a given class of compounds into a fluorescent biosensor. By measuring the binding of different compounds to their cognate biosensor in live cells and comparing these values to those measured in vitro, their cellular uptake and concentrations can be ranked. We demonstrate that our strategy enables the evaluation of the cellular uptake into the cytosol of 2 classes of inhibitors using two different sensor designs; first, sensors comprising the self-labeling protein SNAP conjugated with a chemically modified inhibitor shown for inhibitors of the enzyme human carbonic anhydrase II; and a label-free sensor for inhibitors of protein-protein interactions demonstrated for the protein pair p53-HDM2.

A critical assessment of the synthesis and biological activity of p53/human double minute 2-stapled peptide inhibitors.

BACKGROUND AND PURPOSE: Helix stapling enhances the activity of peptides that interact with a target protein in a helical conformation. These staples are also supposed to change the pharmacokinetics of the molecules and promote cytoplasmic targeting. We assessed the extent to which the pharmacokinetic characteristics are a function of the staple for a peptide inhibiting the interaction of p53 with the human double minute 2 (Hdm2) protein and differ from those of the standard cationic cell-penetrating peptide nona-arginine. EXPERIMENTAL APPROACH: Stapled peptides and linear counterparts were synthesized in free and fluorescently labelled forms. Activity was determined in biochemical time-resolved Förster resonance energy transfer experiments and cellular high-content assays. Cellular uptake and intracellular trafficking were visualized by confocal microscopy. KEY RESULTS: Peptides showed sub-nanomolar potency. For short-time incubation, uptake efficiencies for the stapled and linear peptides were similar and both were taken up less efficiently than nona-arginine. Only for SJSA-1 cells expressing the Hdm2 target protein, the stapled peptides showed an enhanced cytoplasmic and nuclear accumulation after long-term incubation. This was also observed for the linear counterparts, albeit to a lesser degree. For HeLa cells, which lack target expression, no such accumulation was observed. CONCLUSION AND IMPLICATIONS: Cytosolic and nuclear accumulation was not an intrinsic property of the stapled peptide, but resulted from capture by the target Hdm2 after endo-lysosomal release. Considering the rather poor uptake of stapled peptides, further development should focus on increasing the efficiency of uptake of these peptides.

Bioorthogonal Probes for the Study of MDM2-p53 Inhibitors in Cells and Development of High-Content Screening Assays for Drug Discovery.

To study the behavior of MDM2-p53 inhibitors in a disease-relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high-content screening assays. By using automated image analysis with single-cell resolution, we could visualize the intracellular target binding of compounds by co-localization and quantify target upregulation upon MDM2-p53 inhibition in an osteosarcoma model. Additionally, we developed a high-throughput assay to quantify target occupancy of non-tagged MDM2-p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications.

Modifying peptides to enhance permeability.

Recently, peptides have been validated to address intracellular targets and/or to be orally bioavailable. This review describes some of these scaffolds, offers insight in new cyclization methodologies thought to be beneficial to enhance permeability, and highlights modification on peptides thought to improve oral bioavailability. In this context, side chains and back-bone derivatization beneficial to encourage cellular uptake are presented. In addition, new methodologies supporting the assessment of permeability are discussed.

Towards intracellular delivery of peptides.

In view of our more comprehensive understanding with respect to intracellular pathways and their regulation, a vast number of interesting drug targets appear to be in a space that can be addressed neither by small molecules nor by biologics. Especially, interference with intracellular protein-protein interactions, if successful, seems to offer considerable opportunities to expand the application of peptides as therapeutics, in particular in the field of oncology. This review focuses on requirements for the development of peptide therapeutics aiming at intracellular targets. In addition, an outlook for developments in this field based on recent examples for peptides active in cellular assays, highlighting key requirement to assess permeability, is provided.

Synthesis and application of an auxiliary group for chemical ligation at the X-gly site.

An efficient synthesis of an auxiliary group, the 2-mercapto-4,5-dimethoxybenzyl (Dmmb) moiety, to form a Gly-building block is presented. The building block was incorporated into peptides to study the reaction with thiobenzyl-activated derivatives. The target peptides have been obtained by standard chemical ligation reaction, followed by TMSBr-assisted cleavage to remove the auxiliary group. Prior to Dmmb removal, under acidic conditions an unexpected rearrangement was observed and evidence for a mechanism is provided.