Functionalized Resins for the Synthesis of Peptide Alcohols.

Peptide alcohols are clinically important compounds that are underexplored in structure-activity relationship (SAR) studies in drug discovery. One reason for this underutilization is that current syntheses are laborious and time consuming. Herein, we describe the preparation and utility of Rink, Ramage, and Sieber-chloride resins, which enables the use of a general, easy and practical method for the attachment of fluorenylmethoxycarbonyl (Fmoc)-amino alcohols to a solid support, in the synthesis of peptide alcohols. This method is the first straightforward Fmoc/tBu synthesis of peptide alcohols starting from a pre-loaded resin. The synthesized peptide alcohols can be detached from the linkers through conventional methods. Treatment with trifluoroacetic acid (TFA) (95 %) and scavengers such as triisopropylsilane and water for 2 h is sufficient to obtain a fully deprotected peptide alcohol, while treatment with 20 % hexafluoroisopropanol in dichloromethane renders a fully protected peptide alcohol that can be further modified at the C-terminus. As examples, the new resins were used in straightforward, relatively rapid syntheses of the peptide alcohols octreotide, alamethicin, and a segment of trichogin GA IV, as well as the first synthesis of stapled peptide alcohols.

NanoClick: A High Throughput, Target-Agnostic Peptide Cell Permeability Assay.

Macrocyclic peptides open new opportunities to target intracellular protein-protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure-activity-permeability relationships in the optimization of functionally active molecules.

Methods and Approaches for the Solid-Phase Synthesis of Peptide Alcohols.

Many methods have been developed for attaching an alcohol functionality to a solid support. However, not all of these methods are used to obtain peptide alcohols. In this Minireview, we will discuss several of the most important methods and approaches for the synthesis of peptide alcohols and the attachment of hydroxy groups to a solid support for the synthesis of cyclic peptides. Some of the methods include the use of functionalized Wang resin and the attachment of an alcohol to an enol ether resin. We also discuss the use of the chlorotrityl resin, one of the most common linkers used to obtain peptide alcohols. In addition, we outline the recently developed resins with the Rink, Ramage and Sieber handles. The majority of these methods have been used to synthesize many important drugs, such as octreotide and the antibiotic peptaibols.

De-risking Drug Discovery of Intracellular Targeting Peptides: Screening Strategies to Eliminate False-Positive Hits.

Nonspecific promiscuous compounds can mislead researchers and waste significant resources. This phenomenon, though well-documented for small molecules, has not been widely explored for the peptide modality. Here we demonstrate that two purported peptide-based KRas inhibitors, SAH-SOS1 A and cyclorasin 9A5, exemplify false-positive molecules-in terms of both their binding affinities and cellular activities. Through multiple gold-standard biophysical techniques, we unambiguously show that both peptides lack specific binding to KRas and instead induce protein unfolding. Although these peptides inhibited cellular proliferation, the activities appeared to be off-target on the basis of a counterscreen with KRas-independent cell lines. We further demonstrate that their cellular activities are derived from membrane disruption. Accordingly, we propose that to de-risk false-positive molecules, orthogonal binding assays and cellular counterscreens are indispensable.

A Patient-Derived Cell Atlas Informs Precision Targeting of Glioblastoma.

Glioblastoma (GBM) is a malignant brain tumor with few therapeutic options. The disease presents with a complex spectrum of genomic aberrations, but the pharmacological consequences of these aberrations are partly unknown. Here, we report an integrated pharmacogenomic analysis of 100 patient-derived GBM cell cultures from the human glioma cell culture (HGCC) cohort. Exploring 1,544 drugs, we find that GBM has two main pharmacological subgroups, marked by differential response to proteasome inhibitors and mutually exclusive aberrations in TP53 and CDKN2A/B. We confirm this trend in cell and in xenotransplantation models, and identify both Bcl-2 family inhibitors and p53 activators as potentiators of proteasome inhibitors in GBM cells. We can further predict the responses of individual cell cultures to several existing drug classes, presenting opportunities for drug repurposing and design of stratified trials. Our functionally profiled biobank provides a valuable resource for the discovery of new treatments for GBM.

Macrocyclization of an all-d linear α-helical peptide imparts cellular permeability.

Peptide-based molecules hold great potential as targeted inhibitors of intracellular protein-protein interactions (PPIs). Indeed, the vast diversity of chemical space conferred through their primary, secondary and tertiary structures allows these molecules to be applied to targets that are typically deemed intractable via small molecules. However, the development of peptide therapeutics has been hindered by their limited conformational stability, proteolytic sensitivity and cell permeability. Several contemporary peptide design strategies are aimed at addressing these issues. Strategic macrocyclization through optimally placed chemical braces such as olefinic hydrocarbon crosslinks, commonly referred to as staples, may improve peptide properties by (i) restricting conformational freedom to improve target affinities, (ii) improving proteolytic resistance, and (iii) enhancing cell permeability. As a second strategy, molecules constructed entirely from d-amino acids are hyper-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Since neither approach is a complete solution, we have combined these strategies to identify the first examples of all-d α-helical stapled and stitched peptides. As a template, we used a recently reported all d-linear peptide that is a potent inhibitor of the p53-Mdm2 interaction, but is devoid of cellular activity. To design both stapled and stitched all-d-peptide analogues, we used computational modelling to predict optimal staple placement. The resultant novel macrocyclic all d-peptide was determined to exhibit increased α-helicity, improved target binding, complete proteolytic stability and, most notably, cellular activity.

Stereoisomerism of stapled peptide inhibitors of the p53-Mdm2 interaction: an assessment of synthetic strategies and activity profiles.

All-hydrocarbon, i, i+7 stapled peptide inhibitors of the p53-Mdm2 interaction have emerged as promising new leads for cancer therapy. Typical chemical synthesis via olefin metathesis results in the formation of both E- and Z-isomers, an observation that is rarely disclosed but may be of importance in targeting PPI. In this study, we evaluated the effect of staple geometry on the biological activity of five p53-reactivating peptides. We also present strategies for the modulation of the E/Z ratio and attainment of the hydrogenated adduct through repurposing of the metathesis catalyst.