Stapled peptides inhibitors Ali, Amina

University of Groningen

Stapled peptides inhibitors Ali, Amina

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2019

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Ali, A. (2019). Stapled peptides inhibitors: A new window for target drug discovery. University of Groningen.

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Summary (English)

155 Summary

Over the past century, the understanding of any human disease has been linked to the relevant cellular processes, based on molecular insights. As a result, studies of drug target have been conducted in both dry and wet labs from experimental designs to target identification and validation steps. These studies revealed the core role of protein-protein interaction (PPI) in human disease progress making them very interesting targets for modulation with bioactive molecules or inhibitors in order to improve our prospects of developing therapeutic agents.

Examination of the interactions between the validated bioactive molecules and their targets has encouraged tremendous progress in the development of therapeutic agents and expanded their use as PPI inhibitors. Most of these agents belong to two main drug classes: small molecules and biologics. Small molecule compounds have the ability to penetrate the cell membrane to reach their intracellular targets and proved their efficacy – usually to exploit hydrophobic pockets on their targets that are suitable for binding. However, shallow and large protein surfaces are not accessible with small molecules, which fostered the emergence of another therapeutic class - so-called biologics. This class comprises engineered antibodies, growth factors, and other bioactive protein that are typically >5000 Da in size. Biologics are potent and selective binders to their target protein surface, but their use is generally limited to extracellular hits and they have poor oral availability.

As a consequence, a number of challenging intracellular targets are not approachable by small molecules or biologics and have been therefore termed “undruggable”.

Recently, a new class of therapeutic agents engaged in targeting and inhibiting intracellular PPI that are beyond the reach of small molecules and biologics has emerged, ie. peptides. Peptides bridge the gap between small molecules and biologics; moreover, they retain excellent surface recognition to the target surface and minimal toxicity. However, peptides suffer from proteolytic instability and low cell permeability that are mainly associated with their flexible conformation when free in solution. All-hydrocarbon stapling of the helical peptides provides an opportunity to stabilize the bioactive confirmation of the peptides, protect from proteolysis and enhance their drug-like properties and target affinity.

Many chemical strategies have been developed to stabilize α-helix peptides including α- methylation, N-capping, and side-chain-to-side-chain cross-linking. Among the various approaches, hydrocarbon stapling has proven particularly successful, providing numerous examples of extra- and intracellular PPI inhibitors. The reader is directed to Chapter 1 for a general review on PPI stapled peptides inhibitors. The hydrocarbon stapling was applied in our research PPI target, p53- MDM2, as one of the strategies to treat cancer and discussed in detail in Chapter 2 of this thesis.

We have introduced multicomponent reaction (MCR) technique as a potent method in staple

synthesis that were linked to p53-based α-helix peptides at i,i+4 stapling position. Furthermore, we

used the microscale thermophoresis (MST) technique to investigate their binding affinities to our

target protein, being the first to apply this new technology on MDM2 and stapled peptides. As a

result of our study, we have solved three co-crystal structures of these stapled peptides in complex

with a mutant MDM2 to reveal their mode of binding with the hydrophobic cleft; these structures

are considered to be novel.

Furthermore, in this thesis we solved Pex4p:Pex22p complex structure as a novel PPI from the yeast Hansenula polymorpha to elucidate the complex role in peroxisomal recycling. Since peroxisomes are major cellular compartment of eukaryotic cells and are involved in a variety of metabolic functions and pathways; mutation in one of the main recycling peroxins proteins are linked to human peroxisome biogenesis disorder including Pex1, Pex6, Pex10, and Pex4:Pex22 complex.

PPIs rely on two protein partners that interact at specific surface area. The topology of these surfaces must be preserved while protein expression within E.coli cells specially if they are expressed as insoluble protein in bacterial inclusion bodies (IBs). This was the case with our target proteins, the WT- and the mutant human MDM2. For that we developed a system for the refolding buffers screening in order to determine the correct refolding conditions in reliable and time saving manner using the differential scanning fluorimetry (DSF) as an analytical method to detect the correctly refolded proteins. By applying this systematic buffer screen on MDM2 protein that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen, we were successfully able to get stable and properly refolded protein in correct buffer system, pH and essential additives. This approach also allowed the production of MDM2 for structural and biophysical studies.

Chapter 1: Protein-protein interaction (PPI) is a hot topic in clinical research due to the fact that protein networking has a major and primary impact in human diseases development. Such PPIs could be drugs target, thus the need to inhibit or block specific interactions by a molecule has remarkable therapeutic value. Small molecules inhibitors have made some success and already reached clinical trials, notably, p53-MDM2 PPIs that have been studied and explored extensively.

However, small molecules failed to overcome the nature of protein surfaces that are flat and large

and “undruggable” by this class of inhibitor. Instead, biologics were developed as larger inhibitors

to cover PPIs surface and they successfully target PPIs located outside the cell. But due to their size,

biologics have low bioavailability and could be administrated only by injections; furthermore, they

cannot reach intracellular target interfaces. As a consequence, considerable interest has arisen in

next-generation targeting molecules, which combine the target recognition capabilities of biologics

with the cell- penetrating ability of small molecules. A novel class has shown promise in early-stage

studies namely, hydrocarbon-stapled α-helical peptides, which are synthetic mini-proteins locked

into their bioactive α -helical secondary structure through site-specific introduction of a chemical

brace. Stapled peptides show an ability to inhibit intracellular PPIs that previously have been

intractable to inhibition with traditional small molecule or protein-based therapeutics, suggesting

that this class of inhibitors may define a novel therapeutic modality. In this review, we will

highlight what stapling adds to natural- mimicking peptides, describe the revolution of synthetic

chemistry techniques and how current drug discovery approaches have been adapted to stabilize

active peptide conformations, including RCM, lactamisation, cycloadditions and reversible

reactions. An overview on the available RCSB protein data bank stapled peptide high-resolution

structures in complex with their target protein surface, with four selected structures discussed in

details in which the staple makes remarkable interactions with the target surface. All together this

makes stapled peptides promising drug candidates and opening the doors for peptide therapeutics to

reach the currently “undruggable” space.

157 Chapter 2: As an approach to block the interaction between MDM2 and the WT-p53 in cancer cells, different p53-based stapled peptides were synthesized and their staples were linked to the peptide helix at i,i+4 spacing position. The synthesis of the peptides staple was achieved by MCR.

We were successfully able to determine the binding affinities of these stapled peptides toward our target protein MDM2 using MST, and the K

values are measured in the nanomole range. In addition to the potent binding of these stapled peptides, we successfully solved three high- resolution structures of three stapled peptides in complex with MDM2 (L33E). The novel structures revealed a significant interaction between the meta substitution benzene ring in the staple of two peptides (GAR300-Am and Gm) and the hydrophobic pocket of MDM2 by forming a T-shaped π-π interaction with the side-chain of Phe55 residue in MDM2 pocket. Our study supports the notion that stapling reinforces the helicity of the peptides and improves their binding toward MDM2 pocket. Additionally, the staple itself could contribute in the peptide binding by forming an hydrophobic interaction with the target surface.

Phe55

Asp27

Chapter 3: The protein p53 is engaged in the repair of DNA mutations and elimination of heavily damaged cells, thus providing anti-cancer protection. Dysregulation of p53 activity is a crucial step in carcinogenesis. This dysregulation can be achieved either by loss-of-function mutations in p53- encoding gene, or by the overexpression of negative regulators of p53, among which MDM2 is the most prominent one. Antagonizing MDM2 with small molecules restores the activity of p53 in p53- wild type (wt) cells and provides positive outcomes in the treatment of p53-wt cancers.

Previously we have reported the discovery and SAR of a panel of fluoro-substituted indole-based

compounds that bound to MDM2 and inhibited its interaction with p53. Here, we demonstrate the

biological activity and stereoselectivity of the most active compound from this series. Both

enantiomers of the ester form of the compound (5), as well as its corresponding carboxylic acids (6)

were found active in fluorescence polarization (FP), nuclear magnetic resonance (NMR) and

microscale thermophoresis (MST) assays with K

and K

values below or close to 1 µM. However,

only the esterified enantiomer (R)-5a was active in cells, which was evidenced by the activation of

p53, the induction of cell cycle arrest and selective inhibition of the growth of p53-wt U-2 OS and

SJSA-1, but to a lower extent p53-del SAOS-2 cells. The analysis of the crystal structure of MDM2

in complex with the compound (R)-6a revealed the classical three-finger binding mode. Altogether,

the data demonstrate the activity of the compounds and provide the structural basis for further

optimization.

159 Chapter 4: Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein.

Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches

and is thus a time-consuming process. In this study, we use the latest developments in the

differential scanning fluorimetry guided refolding approach as an analytical method to detect

correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary

pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates

that this approach could be applied for determining refolding conditions for several proteins. In

addition, it revealed which “helper” molecules, such as arginine and additives are essential. Four

different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding

approach. Our systematic protocol evaluates the impact of the “helper” molecules, the pH, buffer

system and time on the protein refolding process in a high-throughput fashion. Finally, we

demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors

for improving refolding efficiency.

Chapter 5: Peroxisomes are a major cellular compartment of eukaryotic cells. Peroxisomes are involved in a variety of metabolic functions and pathways according to species, cell types and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode Peroxin proteins. Peroxisomal membranes proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally.

Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, releasing of the cargo into the lumen and recycling involve a number of Peroxins, but a key player is the Pex4p:Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p:Pex22p complex is essential for monoubiquitination at the conserved Cysteine residue of Pex5p. Absence of the Pex4p:Pex22p inhibits Pex5p recycling and hence PTS1 protein import. In this paper we report the crystallization of Pex4p and the Pex4p:Pex22p complex from the yeast Hansenula polymorpha and data collection of the complex crystals protein structure at 2.0 and 2.85 Å resolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanisms of the Pex4p:Pex22p complex and its role in peroxisome biogenesis.

Pex4

Pex22

161 Chapter 6: Pex4p is a peroxisomal E2 involved in ubiquitinating the conserved cysteine residue of the cycling re- ceptor protein Pex5p. Previously, we demonstrated that Pex4p from the yeast Saccharomyces cerevisiae binds directly to the peroxisomal membrane protein Pex22p and that this interaction is vital for receptor ubiquitination. In addition, Pex22p binding allows Pex4p to specifically produce lysine 48 linked ubiquitin chains in vitro through an unknown mechanism.

This activity is likely to play a role in targeting peroxisomal proteins for proteasomal degradation.

Here we present the crystal structures of Pex4p alone and in complex with Pex22p from the yeast Hansenula polymorpha. Comparison of the two structures demonstrates significant differences to the active site of Pex4p upon Pex22p binding while molecular dynamics simulations suggest that Pex22p binding facilitates active site remodelling of Pex4p through an allosteric mechanism.

Taken together, our data provide insights into how Pex22p binding allows Pex4p to build K48-

linked Ub chains.