University of Groningen
Stapled peptides inhibitors Ali, Amina
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Chapter 2
Novel Crystal Structures Of Stapled Peptides In Complex
With Mutant MDM2 (L33E): A New Approach Toward
Non-Genotoxic Anti-Cancer Drugs
Ameena M. Ali, Jack Atmaj, Niels van Oosterwijk, Daniel G. Rivera, Matthew R. Groves, and
Alexander Dömling (Manuscript in preparation)
1 Introduction
More than 50% of all human cancers are triggered by functional inactivation of p53, which resulted from either mutation or a deletion in p53 gene, called TP53 [1–3]. This high prevalence of p53-related cancers is consistent with the biological function of p53 as a main tumor suppressor. p53 plays a central role in many cellular processes including cell cycle arrest, apoptosis, angiogenesis, senescence and cancer suppression [4–8]. Although the remaining 50% of cancers harbor wild-type p53 (WT-p53), its transactivation activity is inhibited by MDM2 overexpression, which is known to be the main negative regulator for p53 within cells [9,10]. Analysis of human tumor samples from 28 different types of human cancers showed that in 7% of human cancers MDM2 gene is either amplified or overexpressed by p53 and mutations [11,12]. These data correlate MDM2 overexpression with the poor function of p53 as tumor suppressor and indicate that MDM2 is the chief primary regulator of p53 function [12–15].
MDM2 (murine double minute 2) is a human oncoprotein that has a synergetic function with p53 in controlling the cell cycle and preventing the conversion of normal cells to cancerous ones [7]. Cellular levels of p53 and MDM2 are controlled via a negative feedback loop. As MDM2 is the transcriptional product of TP53-gene induction, in turn, MDM2 regulates the level and growth-suppressive function of p53 in several ways [16,17]. In unstressed cells, MDM2 controls p53 negatively through one of two distinctive mechanisms, either by 1) direct protein-protein interaction (PPI) or 2) targeting p53 for proteasome-mediated degradation via its E3 ubiquitin ligase activity [18–20]. Three key hydrophobic residues in p53 helix contributing to MDM2 binding: Trp23, Leu26 and Phe19; as reported in the first co-crystal structure of MDM2 in complex with a 15-residues p53 peptide (15-29) (PDB code 1YCR) [21]. As a result, this mode of binding between MDM2-p53 is known as the three-finger pharmacophore model. Since then, this binding model is now widely accepted and was used to design several potential drug compounds like small molecules and peptides towards the MDM2:p53 hydrophobic interface [22–24]. Nevertheless, an extended four finger model was recently proposed and well described in a co-crystal structures of MDM2 N-terminus in complex with a small molecule inhibitor YH-300 (PDB 4MDN) [25–27]. Based on the structural knowledge obtained from these co-crystal structures, blocking the PPI between p53 and MDM2 has been proposed as promising non-genotoxic therapeutic strategy in the treatment of cancer.
The basic idea behind designing p53-MDM2 inhibitors is to mimic the binding interface of p53 α-helix (transactivation domain, residues 15 to 29) to the buried, hydrophobic and well-structured MDM2 cleft (N-terminal domain, residues 17 to 125) that has a surface area of ~700Å2. Several small molecules inhibitors have been synthesized and shown to block p53−MDM2 PPI, and some of them have reached early clinical trials [28,29]. The most well studied p53-MDM2 inhibitor from this class is Nutlin and its derivatives produced by Hoffmann-La Roche in 2004. Nutlin-2 was the first described co-crystal structure of a small molecule in complex with MDM2 (residues 25-108) (PDB 1RV1; [4]), whereas, Nutlin-3a has been extensively tested on several human cancer cell lines and showed a high capacity to activate WT-p53 and induce selective apoptosis in tumor cells
only [4,15]. Another Nutlin-based compound (RG7112) completed Phase I clinical trials and was tested on patients with advanced solid and soft-tissue tumors and hematological malignancies [30]. The success of Nutlin-based and other small molecules to interrupt the MDM2-p53 interaction was clear, and promising outcomes were found when tested on different cancers as described in the previous section. However, regardless of their high oral bioavailability and cellular diffusion, these small entities cannot cover the huge surface of the most druggable PPI interfaces of 1500-3000Å2. Furthermore, these surfaces are dynamic having different conformations and amino acid residue composition depending on the partner binding protein, particularly the hot spots residues that are essential for binding [31–36]. All of these limitations created the necessity for a new class of inhibitors with higher affinity and specificity toward MDM2, as well as improved physiochemical and pharmacological properties, which is linked to the objective of our recent work in this chapter. Subsequently, intensive research efforts give the opportunity for protein-based scaffolds (peptides) to disrupt PPIs to be promising, potent and selective inhibitors with distinct chemical structures. Historically, peptides and their derivatives were first synthesized in 2007 based on phage display libraries, consisting of a 12-residues peptide (pDI) and its 11 derivatives that have a high activity toward MDM2 and its homolog MDMX (known also as MDM4) [37,38]. Later, more specific peptide inhibitors were designed based on the p53 peptide sequence and showed higher binding affinities toward MDM2, notwithstanding of their low cellular permeability and poor stability against proteolytic degradation.
To overcome these limitations, all-hydrocarbon stapled-peptides were introduced; in which the bioactive α-helix peptide is locked by a hydrocarbon brace through a specific site within the α-helix sequence, an all-hydrocarbon staple [32]. Stapling greatly improves the pharmacologic performance of peptides, enhances their affinity to the target, increases their proteolytic resistance, and improves their penetration into the cell through endocytic vesicle trafficking [29,32]. Recently, several staple peptides with effective MDM2 inhibition and higher affinity up to picomoles (pM) related to the native p53 peptide (600 nM, [39]) have been discovered. The best example here is the SAH-p53–8 stapled peptide, derived from the helix sequence of the WT-p53 (PDB code: 3V3B, [40]). This stapled peptide was found to inhibit MDM2 and MDMX interactions with p53 in both in vivo and
ex vivo experiments, in addition to the protease resistance - combined with increased cellular uptake
due to the presence of stapling. Significantly, SAH-p53-8 has efficiently induced a tumor-suppressive response in vivo. A unique feature found in SAH-p53-8 and shared with another stapled peptide, named E1 (PDB 5AFG, [41]), is that the staple itself made several hydrophobic interactions with the same residues on MDM2 surface (Leu54, Phe55, Gly58, and Met62). In addition to these later unique stapled peptides, others have also shown strong binding, stability and
impact on the peptide binding either directly via hydrophobic interactions with the target surface or indirectly by reinforcing the secondary structure of α-helix peptide in order to increase the stability of its binding-mode. As a consequence, to reactivate WT-p53 and induce apoptosis in tumor cells, extensive optimizations for this new scaffold are needed to generate more stable, highly soluble and cell permeable drug-like agents with increased oral bioavailability. In this paper we report three novel co-crystal structures of staple peptides in complex with mutant MDM2 (L33E). Our ongoing endeavor to discover p53-MDM2 PPI inhibitors based on the structural and crystallographic data provided from our high-resolution structures will open a new window of discovery to overcome the poor clinical and pharmacokinetic properties of the current agents in clinical trials.
2 Methods
2.1 MDM2 (L33E) mutant gene design and cloning
MDM2 construct corresponding to the N-terminus domain and the hydrophobic-pocket region of MDM2 protein (Glu24-Val109) with leucine 33 mutated to glutamic acid residue was designed using SnapGene Viewer (4.1.9). The designed construct was codon-optimized for E.coli and ordered from Eurofins, Germany. The sequence of previously published structure of the same mutant protein (PDB 1RV1) was used as reference to design our construct. Subsequently, the gene was cloned into pETM-13 expression vector (EMBL, Germany) between the NcoI and BamHI restriction sites. Both the gene and the vector were double digested with the respective restriction enzymes, gel purified - followed by 4hrs ligation at 37 °C. Finally, the construct was verified by sequencing before protein expression. The details of the expression construct are summarized in Table (1).
Table (1)
MDM2 (L33E) construct information
Source organism Homo sapiens
Forward/ Reverse primer N/A
DNA source Synthetic codon-optimized gene (Eurofins)
Cloning Vector pXE-A128
Expression Vector pETM-13
Expression host Escherichia coli
Complete amino acids sequence of the construct produced
METLVRPKPELLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRLYDEKQQHI VYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVV
The amino acid in red indicate the position of the L33E mutation
2.2 MDM2 (L33E) protein production:
The expression plasmid containing MDM2 (L33E)/pETM-13 was transformed into E. coli BL21* (DE3) cells for protein expression. A single colony was selected for and inoculated into a 50ml pre-culture LB-enriched media supplemented with 35 µg/l of kanamycin and chloramphenicol. This culture was incubated overnight at 30°C while shaking at 180 rpm. The overnight-culture was inoculated into the main culture (1L) then allowed to grow at 37°C until an OD600 of 0.6-0.8 was reached, at which point protein overexpression was induced by the addition of 1 mM
isopropyl-β-D-1-thiogalactopyranoside (IPTG). After induction, the culture was incubated with shaking for a further 5hrs at the same temperature. MDM2 (L33E) was expressed as insoluble protein within inclusion bodies (IBs), the cells pellet was collected by centrifugation using a F12-6x500 LEX rotor (Thermo Scientific) and re-suspended in 30 ml PBS buffer, pH7.4 containing 10m M dithiothreitol (DTT). Purification of mutant MDM2 protein was initiated by sonicating the re-suspended pellet in PBS buffer, pH7.4, 10m M DTT supplemented with protease- inhibitor cocktail tablets (Protease Inhibitor Cocktail Set II, MERK) for 10 min at 30% sonication power (Branson 250 Digital Sonifier). The lysate was cleared by centrifugation using F20-12x50 LEX rotor (Thermo Scientific) at 12,000 rpm at 4°C; samples were collected for both the supernatant and the pellet for SDS-PAGE gel analysis. The collected IBs were washed three times: twice with the washing buffer (PBS pH7.4, 10m M DTT, 0.05% Triton) and the third wash with PBS buffer, 10mM DTT without Triton at pH7.4.
The protein was then denatured in a buffer of 6 M Guanidine-hydrochloride, 100 mM Tris pH 8, 1 mM EDTA and 10 mM β-mercaptoethanol (BME). Dialysis was conducted against 4 M guanidine hydrochloride pH 8 supplemented with 10 mM BME. Next, the mutant MDM2 was refolded by shock dilution at 1:100 ratio of protein to refolding buffer followed by overnight slow mixing at 4°C. All purification steps were performed with ice-cold buffers. A hydrophobic interaction chromatography resin (HIC) was applied to the refolded protein (Butyl-Sepharose 4 Fast Flow (GE Healthcare Life Sciences)) as an intermediate polishing step. For this, the protein was incubated with 1.5 M of ammonium sulfate for 2hrs at 4°C and the supernatant was collected with centrifugation at 10,000 g, then carefully collected and incubated for another 2hrs with 2.5 ml of pre-equilibrated HIC beads with Equilibration/Wash buffer (10 mM Tris pH7, 1 mM EDTA, 10 mM DTT and 1.5 M Ammonium sulfate) at 4°C. Subsequently, the resin was collected and washed with four times the bed volume of Equilibration/Wash buffer; followed by protein elution in Elusion buffer consisting of 100m M Tris pH7.2 and 5 mM DTT. Fractions containing MDM2 were pooled, concentrated and then loaded onto a Superdex 75 16/60 column equilibrated with 20 mM HEPES pH7, 50 mM NaCl and 5 mM DTT. Mutant MDM2 eluted in a single peak and was homogeneous, as judged by 15% SDS–PAGE gel.
2.3 All-hydrocarbon stapled peptides synthesis
The all-hydrocarbon stapled peptides were gratefully provided by our collaborator Dr. Daniel Rivera (Faculty of Chemistry, University of Havana, Cuba). The peptides design was based on the native p53 sequence following Solid Phase Peptide Synthesis (SPPS) method. The synthesis of the staple was based on multicomponent reaction (MCR) and linked to the helix at i,i+4 stapling position. The peptide chemical structure, sequence and molecular weight are listed in Table 2.
Table 2
The details of the synthetic all-hydrocarbon stapled peptides
Peptide Chemical structure M.W (Da)
GAR300-Gp 1938,9628
GAR300-Am 1910,9315
GAR300-Gm 1938,9628
2.4 Co-crystallization of MDM2 (L33E) with stapled peptides
The purified MDM2 was concentrated up to 8 mg/ml using Vivaspin TURBO 4R ultrafiltration column (5000 MWCO-Sartorius) and the concentration was determined by UV absorbance at 280 nm using an extinction coefficient value of 10,430 M-1cm-1. Three stapled peptides GAR300-Am, GAR300-Gp and GAR300-Gm were co-crystallized with the mutant MDM2. A stock solution of 100 mM was prepared for each peptide in 100% dimethyl sulfoxide (DMSO), then mixed with the protein in a 5:1 ratio and subsequently incubated overnight at 4°C for complex formation. The complex was centrifuged at high-speed 14,000 rpm using a F45-30-11 rotor (Eppendorf) at 4°C to remove any precipitation, followed by concentrating each complex to 20 mg/ml using Amicon Ultra 0.5ml centrifugal filter (3000 MWCO-MERK). The three complexes “MDM2 (L33E)/GAR300-Am, MDM2 (L33E)/GAR300-Gp and MDM2 (L33E)/GAR300-Gm” were subjected to extensive crystallization screening using the sitting-drop vapour-diffusion method and a nanodispenser robot (Gryphon, Art Robbins) in 96-well plates (Polystyrene MRC Crystallization Plates- Molecular Dimensions). More than 500 crystallization conditions were screened from six commercially available screening kits each consisting of 96 conditions (PACT premiere, JCSG-plus, Clear Strategy Screen I and II (Molecular Dimensions), Index and Salt RX (Hampton Research). Promising conditions were further optimized manually by altering protein to buffer ratio within the sitting drop (either 1:1 or 2:1), concentration of precipitation agents and pH. From theses optimized conditions, two successful conditions were selected for each complex. The sitting-drop vapour-diffusion technique was used to optimize these conditions and drops were setup manually by mixing each complex (20 mg/ml) with reservoir buffer at 1:1 ratio and the plates were incubated at 20°C. Several fully-grown crystals of each complex appeared in 2-5 days and were found in different reservoir solutions. The details of the final conditions obtained from optimization experiments are listed in Table 3.
2.5 Data collection and processing
The crystals were mounted in nylon loops and flash-cooled in liquid nitrogen after quick soaking in cryo-protectant solutions that were similar to the crystallization solution with an additional 40% PEG-2000 for MDM2 (L33E)/GAR300-Am complex crystals, 35% PEG-3350 for MDM2 (L33E)/ GAR300-Gp complex crystals, and 50% PEG-400 for MDM2 (L33E)/GAR300-Gm complex crystals. High-resolution diffraction data for the three complexes structures were collected at the PETRA III synchrotron, Hamburg, Germany at a wavelength of 1.0332Å and crystals were shipped using dry-shipping container (Taylor–Wharton). The crystals of MDM2 (L33E)/ GAR300-Am, GAR300-Gp and GAR300-Gm complexes diffracted to 2.09, 1.8 and 2.40 Å, respectively. The images were indexed, integrated and scaled using XDS (Kabsch, 2010), The crystals of MDM2 (L33E)/GAR300-Am and /GAR300-Gp complex belong to P3121 space group, while the crystals of the mutant protein in complex with GAR300-Gm belong to P 6122 space group, determined using
POINTLESS (Evans, 2011) from the CCP4 suite (Winn et al., 2011). Data-collection and
processing statistics are presented in Table 4.
To determine the initial phases for the stapled peptide in complex with mutant MDM2, conventional molecular replacement was performed (rotation, translation and rigid body fitting) using Phaser (McCoy et al., 2007) with the coordinates of human MDM2 comprising the same mutation (Leu33 to Glu) as an initial search model (100% sequence identity for residues 24–109; PDB 1RV1; Vassilev et al., 2004). After the first cycle of refinement, the three stapled peptide helices were clearly visible in the electron density maps. Manual building of the peptide backbone, the cyclic-linker structure and electron density interpretation were performed after each refinement cycle using the program COOT (Emsley et al., 2004). Similarly, the electron density of the staple for both peptides, GAR300-Gp and GAR300-Am was clearly visible in the electron density, however, the linkage GAR300-Gm was not found in density and was modeled using the MOLOC software (Gerber et.al, 1995).
Table 3
Reservoir solutions and crystallization information for the three complexes structures
Complex structure MDM2 (L33E): GAR300-Am MDM2 (L33E): GAR300-Gp MDM2 (L33E): GAR300-Gm
Method Sitting-drop vapour diffusion
Plate type 96-well Polystyrene MRC Crystallization Plate (Molecular Dimensions)
Temperature (C) 20 °C
Protein conc. 20 (mg ml-1)
Buffer composition of
protein solution 20mM HEPES pH7, 50mM NaCl and 5mM DTT
Buffer composition of reservoir solution
0.1M BIS-TRIS pH 6, 0.2M ammonium acetate 30% PEG-3350
0.1M TRIS pH 7.5, 0.2M Ammonium Sulfate, 25% PEG-3350 0.056M Sodium phosphate monobasic monohydrate pH 8.2, 1.344M Potassium phosphate dibasic. 0.1M TRIS pH 8, 0.2M
Trimethyl N-oxide dihydrate, 20% PEG-2000
0.1M TRIS pH 7.5, 0.2M Ammonium Sulfate, 30% PEG-3350
2.1M DL-Malic acid pH 7.0 0.1M TRIS pH 8.5, 0.2M
Trimethyl N-oxide dihydrate, 15% PEG-2000
0.1M TRIS pH 8, 0.2M Ammonium Sulfate, 25% PEG-3350
0.1M TRIS pH 8.5, 0.2M Trimethyl N-oxide dihydrate, 20% PEG-2000
0.1M TRIS pH 8, 0.2M Ammonium Sulfate, 30% PEG-3350
0.1M TRIS pH 9, 0.2M Trimethyl N-oxide dihydrate, 20% PEG-2000
0.1M TRIS pH 8, 0.2M Ammonium Sulfate, 35% PEG-3350
0.1M TRIS pH 8.5, 0.2M Ammonium Sulfate, 25% PEG-3350
0.1M TRIS pH 8.5, 0.2M Ammonium Sulfate, 30% PEG-3350
Volume and ratio of
the drops 0.8 µl (1:1 ratio)
The reservoir solutions in bold indicate the conditions that contained the selected crystals for data collection, refinement and model building of the complex structures and used for the figures in this chapter
2.6 Microscale Thermophoresis (MST) and Kd measurement
The binding affinities of the stapled peptides toward the wild-type MDM2 (WT-MDM2) protein were analyzed using the MST technique. The coding region (11-118) was cloned into bacterial pETM-20 expression vector (kindly provided by prof. Tad A. Holak, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Cracow, Poland) and the production of the wild-type protein was performed as described previously (Czarna et al. 2009; Twarda-Clapa et al. 2017). To obtain fluorescently labeled protein, the standard labeling protocol of NanoTemper Protein Labeling Kit RED-NHS (L001- NanoTemper Technologies, Munich, Germany) was followed. In MST experiments, the concentration of the labeled WT-MDM2 was kept constant (100 nM), while the unlabeled partner peptides were serially diluted in MST buffer (50 mM Tris-HCl buffer, pH 7.6, 150 mM NaCl, 10 mM MgCl2 and 0.05% Tween-20) at 1:1 protein to buffer ratio. Sixteen10 µL samples ranging in concentration from 1 µM to 0.031 nM were incubated for 4 minutes at room temperature before loading each reaction mixture into Premium coated-capillaries (MO-K025, NanoTemper Technologies).
MST measurements were carried out by Monolith NT.115 instrument (NanoTemper Technologies, Germany) and the binding affinities (Kd values) were analyzed in the NanoTemper MO. Affinity Analysis v2.2.4 software, following the low of mass action model to fit the normalized fluorescence signal into a sigmoidal curve, in which the unbound and saturated state form a lower and upper plateau (Seidel et al, 2013).
𝐹𝐵 =[𝐴𝐵] [𝐵] = 𝐴 + 𝐵 + 𝐾!− ( 𝐴 + 𝐵 + 𝐾!)!− 4[𝐴𝐵] 2[𝐵] Where: FB fraction bound
[A] concentration of unlabeled titrated partner
[B] concentration of fluorescent labeled partner that is fixed [AB] concentration of bound complex of A and B
Kd equilibrium dissociation constant
The measurements were carried out at 60% MST power, an LED excitation source with λ = 625 nm at 22 °C with laser off/on times of 5ms.
3 Results
3.1 MDM2 (L33E) mutant gene design and cloning
The cloned MDM2 (L33E) gene and the pETM-13 vector were digested with NcoI and BamHI restriction enzymes for 1 hr at 37 °C followed by 4 hrs of ligation reaction. The resulting products from digestion were separated on a 1% agarose gel in using a 1kb DNA ladder marker (Axygen Bioscience), Figure 1. The successful transformation of E.coli Turbo cells with MDM2 (L33E)/pETM-13 plasmid was confirmed with sequencing and colony PCR using T7 primers. Colonies were selected randomly for colony PCR and the PCR products were analyzed on 1% agarose gel for the presence of the inserted gene in parallel with the negative control (undigested pETM-13 vector) and 1kb DNA ladder marker (Axygen Bioscience) as shown in Figure 2.
M pETM-13 pETM-13 MDM2 (L33E) Uncut Digested Digested
8000 6000
3.2 MDM2 (L33E) protein production
The mutant protein was expressed as insoluble protein within inclusions bodies (IBs). Purification started with the washing of the IBs two times with PBS pH7.4, 10 mM DTT and 0.05% Triton buffer and a third wash with PBS pH7.4 buffer containing 10 mM DTT. Two samples were collected from each washing step, for both the supernatant and the pellet. These samples were examined on a 15% SDS-PAGE gel along with the unstained-protein marker (Thermo Fisher Scientific, US). Protein recovery increased with the washing steps as indicated from the band density on the gel. After refolding and HIC, samples of the proper refolded mutant MDM2 and the polishing step with Butyl-Sepharose medium were analyzed on 15% SDS-PAGE gel for protein purity, Figure 3. The last purification step was conducted using a Superdex 75 16/60 column, where the mutant protein eluted as a single, sharp peak (Figure 4), the collected fractions of the eluted protein were evaluated on 15% SDS-PAGE gel (Figure 3) and the achieved protein purity was >95%. The band of the mutant protein indicated in the figures as a purple arrow with 10 kDa in size.
Figure 2:1% agarose gel of colony PCR reaction from the random selected transformed Turbo cells. The resulted bands of
500 bp in size confirm the presence of the target gene MDM2 (L33E) in addition to the vector-flanking region. The PCR products were run with the undigested pETM-13 vector (1000 bp) as a negative control. M lane is the marker bands in base pair size (bp).
M Neg- C1 C2 C3 C4 C5 C6 Control
1000
500
Figure 3: 15% SDS-PAGE gel electrophoresis for the collected samples from the supernatant and the pellet for each IBs
washing step (Left). Where Lys= Lysate after sonication, LysS= supernatant of the lysate after centrifugation,
W1S/W2S/W3S= supernatant samples after centrifugation of the first, second and third wash, W1P/W2p/W3p= pellet
samples after centrifugation of the first, second and third wash. The molecular weight of MDM2 (L33E) is 10 kDa, indicated with a purple arrow on gel. The refolded protein (Mdm2 Refold), Butyl-sepharose medium washing (W), the eluted protein from beads (E), the concentrated protein loaded on the Superdex 75 column (Mdm2 Conc.), as well as the eluted protein from the same column (S75 E) were judged for homogeneity on SDS-PAGE gel (Right). M for the unstained protein marker in kDa. M Lys LysS W1S W1p W2S W2p W3S W3p 116 35 14.4 ! M Mdm2 Butyl- Sephrose Mdm2 S75 Refold W E Conc. E ! 116 35 14.4
3.3 MDM2 (L33E) co-crystallization with stapled peptides
Initial crystallization screening for the three complexes were conducted using commercially available screening kits and revealed promising conditions for optimizations. Hundreds of small and thin needle-shaped crystals were found for MDM2 (L33E)/GAR300-Am complex in several conditions from the Index (Hampton Research) and PACT premiere (Molecular Dimensions) screening kits. Hundreds of crystals similar in shapes to the previous complex were also found for MDM2 (L33E)/ GAR300-Gp complex in other conditions from the same later kits as well as the Salt Rx (Hampton Research) screening kit. For MDM2 (L33E)/ GAR300-Gm complex, diffraction-quality crystals were found in two screening conditions, one condition from JCSG (Molecular Dimensions) and the second one from Index (Hampton Research) screening kits (Figure 5). Only MDM2 (L33E)/ GAR300-Gm complex crystals were fished from the screening drops and send to the DESY synchrotron facility, Hamburg, Germany.
Figure 4: Chromatogram of the mutant MDM2 from size-exclusion chromatography using a Superdex 75 16/60 column. The
protein peak eluted at 0.88 column volume.
Large diffraction-quality crystals (0.1-0.4 mm) were obtained by manual optimization of the above initial conditions using the sitting-drop vapour-diffusion technique. MDM2 (L33E)/GAR300-Am complex crystals grew in a solution consisting of 0.1 M TRIS pH8, 0.2 M Trimethylamine N-oxide dihydrate, 20 % PEG-2000. These crystals diffracted to 2.09Å resolution (Figure 6a). While, MDM2 (L33E)/GAR300-Gp complex crystals were obtained in 0.1 M TRIS pH8, 0.2 M Ammonium Sulfate, 30% PEG-3350 that diffracted to 1.8Å resolution (Figure 6b).
3.4 Microscale Thermophoresis (MST) and Kd measurement:
WT-MDM2 was successfully expressed in BL21 (DE3) cells, refolded and purified as described previously (Czarna et al. 2009; Twarda-Clapa et al. 2017). The protein purity was established by SDS-PAGE gel electrophoresis as a single thick band on the gel of 13.8 kDa in size (purple arrow, Figure 7a). The protein concentration was calculated by absorbance measurement at 280 nm using 10430 M-1cm-1 as extinction coefficient. The pure protein was subsequently labeled according to the RED-NHS Protein Labeling Kit (L001- NanoTemper Technologies, Munich, Germany). The fluorescent-labeled protein was pre-tested in Monolith NL1.5 for fluorescence signals (ideally ≥300 fluorescence counts) and aggregation. In our case, WT-MDM2 gave a strong fluorescence signal of approximately 415 counts and no aggregation was observed as concluded from MST curve analysis, which showed a straight smooth-line without bumps (Figure 7b). The binding affinities of the staple peptides toward the WT-protein were measured in the Monolith NL1.5 and Kd values were calculated by normalizing the experimental data to the initial fluorescence signal (usually equal to 1) and fitting them to a sigmoidal, s-shaped binding curve. All of the stapled peptides showed high binding affinities to WT-protein with dissociation constants of 31, 2.4 and 3.14 nM for GAR300-Am, GAR300-Gp, and GAR300-Gm respectively. MST curves for all tested stapled peptides are shown in Figure 8.
Figure 6: Photographs of MDM2 (L33E) crystals in sitting-drop vapor-diffusion experiments in complex with a)
GAR300-Am stapled peptide with crystals size of 0.1 mm, and b) GAR300-Gp stapled peptide with crystals size of 0.4 mm. Photographs were taken after 5 days of incubation at 20 °C.
b) a)
b)
Figure 7: a) SDS-PAGE gel showing the fractions collected during the purification of WT-MDM2, and protein band with
13.8 kDa in size is indicated with a purple arrow, M for the unstained protein marker in kDa size b) Pre-test for fluorescently labeled WT-MDM2 post purification. On the left, the capillary scan for the labeled protein fluorescence signals and 415 counts were obtained, on the right, the MST-trace displays a smooth-line (not bumpy), which indicates no aggregations in the protein sample and no adsorption of the labeled protein to the capillary wall.
M Mdm2 Butyl- Sephrose Mdm2 S75 Refold W E Conc. E ! a) 116 35 14.4 a) b)
Figure 8: MST binding curves used to evaluate the binding of stapled peptides to WT-MDM2. The concentration of the
purified, fluorescently labeled WT-MDM2 was kept constant in all reactions (100 nM) and the protein was mixed with increasing concentrations of a) GAR300-Am or b) GAR300-Gp or c) GAR300-Gm ranging from 1 µM to 0.02 nM and an
3.5 Structural properties of constrained stapled peptides bound to mutant MDM2
The three stapled peptides were co-crystallized with mutant MDM2 contained glutamic acid at position 33 instead of leucine for no other reason than enhancing protein crystallization. Each stapled peptide was screened in more than 300 buffer conditions, screening drops were set following sitting- drop vapour-diffusion technique in 96-well plates at 1:1 ratio of the complex to crystallization buffer using a Gryphon robot (Art Robbins). A similar technique and ratio were used to manually setup the optimization drops. The crystals of GAR300-Am and GAR300-Gp stapled peptides in complex with mutant MDM2 were optimized in order to get diffraction-quality crystals, whereas MDM2 (L33E)/GAR300-Gm complex crystals were fished directly from screening drops and further optimization was not required. All co-crystallization plates were incubated at 20 °C and crystals appeared in five days. The resulting three structures are the first reported structure seen thus far with a hydrocarbon constrained peptide in complex with MDM2 (L33E) and may be considered novel.
The co-crystal structure of the three stapled peptides in complex with MDM2 (L33E) were solved to elucidate their binding mode. In each complex structure, one molecule of mutant MDM2 was found in asymmetric unit binding one staple peptide molecule. The peptide helix is located within the deep hydrophobic cleft of MDM2 at the WT-p53 binding site. The α-helix of the three stapled peptides was very clear within the electron density and the complex structures diffracted at high resolution of 1.8, 2.09 and 2.24Å for MDM2 (L33E)/GAR300-Gp, MDM2 (L33E)/GAR300-Am and MDM2 (L33E)/GAR300-Gm, respectively.
When compared to the crystal structure of p53 helix in complex with N-terminal domain of MDM2 (PDB: 1YCR), all the peptides bind to the deep MDM2 groove in a similar way to the WT-p53 peptide, mimicing the conserved p53-derived Mdm2 interaction motif (Phe19, Trp23, Leu26) in an identical orientation (Figure 9a). In addition, a native hydrogen bond was found between the indole N of Trp23 (according to WT-p53 sequence numbering) in the stapled peptide helix and the carbonyl oxygen of Leu54 on MDM2 hydrophobic deep pocket. The H-bond has a distance ranging between 2.9-3.2 Å in the three structures. The same H-bond was found in the native p53 (PDB 1YCR) as well as SAH-p53-8 stapled peptide (PDB 3V3B) structures in complex with MDM2. Tyr100MDM2 that lies in close approach to Pro27 of the WT-p53 makes a rotation toward the C-terminus of the three stapled peptides, specifically with contact to the Ala30 residue with a distance ranging between 3-3.6Å in the three structures (Figure 9c).
Remarkably, the last three residues of GAR300-Gp, Am and Gm do not adopt the linear conformation that is observed at the C-terminus of WT-p53 while binding MDM2 cleft; instead they continue the helical fold to create a coiled α-helix covering residues 19−30 in the WT-p53 sequence (Figure 9b). This feature was also found in other reported structures of MDM2 in complex with SAH-p53-8 (PDB 3V3B), YS-1 (PDB 4UE1) and YS-2 (PDB 4UD7) stapled peptides.
Figure 9: a) The three finger pharmacophore binding mode of the three stapled peptides helix GAR300-Gp (orange), GAR300-Am
(green) and GAR300-Gm (purple) that is identical to the native p53 helix (yellow) binding to MDM2 hydrophobic cleft (PDB 1YCR). b) The stapling of the peptides preserves and reinforces the coiled helical structure extending even beyond Leu26 toward the C-terminal, which is not the case with the WT-p53 helix that adopt a linear confirmation instead. c) Tyr100 in MDM2 pocket makes a closer contact with the C-terminus of three stapled peptides (cyan line), this phenomena is common in most MDM2 structures 3V3B, 4UD7 and 4UE1 in relative to the WT-p53 structure.
Interestingly, the benzene ring in GAR300-Am staple itself makes a perpendicular (T-shaped) π-π interaction and was found between the benzene ring of the stapled and MDM2 Phe55 residue side-chain, moreover, the same residue form a Van der Waal interaction with the staple carbon, which located at the side where the staple linked to the peptide helix by Asp27. A third hydrophobic interaction was found internally within the stapled peptide, between the stapled Cα and Asp27 C in the peptide helix. These interactions could be related to the meta substitution at the benzene ring in the staple, allowing the staple to form quadrupole/quadrupole interactions with Phe55 side-chain ring at a separation distance equal to 3.7Å (Figure 10a).
a) Phe19 Trp2 3 Leu26 b) Leu26 c) Tyr100
MDM2 surface, which is also the case of GAR300-Gm staple. No other hydrophobic interactions were found between the staple and the target protein surface.
Figure 10: The intimate interactions of the a) GAR300-Am and b) GAR300-Gm staples with MDM2 surface at the hydrophobic
cleft. Both staples have a benzene ring at meta substitution position. The Van der Waal interactions are in yellow dash-lines and the involved Phe55 residue in T-shaped π-π interaction shown as stick, while water molecules as red spheres.
The highest resolution structure (1.8Å) of GAR300-Gp in complex with MDM2 mutant disclosed an α-helical structure with the triad residues Phe19, Trp23 and Leu26 occupying their consensus pockets, in addition, to the native H-bond between the N atom of Trp23 indole ring and the carbonyl oxygen of MDM2 Leu54 residue as mentioned above. Additional two H-bonds were formed between the N atoms of Phe19 and Thr18 in the peptide helix and the carbonyl oxygen of Gln72MDM2 residue (Figure 11a). More hydrophobic interactions were found also between GAR300-Gp helix and MDM2 nonpolar surface involving Gly58MDM2, Ile61MDM2, His73MDM2, Val93MDM2, His96MDM2 with Phe19GAR300-Gp and Trp23GAR300-Gp, Tyr22GAR300-Gp, Leu26GAR300-Gp respectively(Figure 11b and c). The staple was solvent-exposed and makes no direct interaction with the protein surface. This could be influenced by three factors: 1) the staple length, which is longer than the other two peptides with a two-carbon link in the peptide helix at position 4 and 11 instead of one carbon, which is the case in GAE300-Am and Gm. This makes the staple confine itself away from MDM2 pocket toward the solvent side. 2) Structural analysis of the area around GAR300-Gp staple using COOT confirms that crystal packing effects could be a logic reason to prevent staple-protein interaction, and the staple found to be located at the interface of another protein molecule within the crystal lattice. 3) The staple consist of a para substitution at the benzene ring might not interfere with any interaction with the protein surface in contrast to the meta substitution in GAR300-Am and Gm, which favor T-shaped π-π interaction.
a) Phe55 Asp27 Phe55 b) Lys51 Gln59
Figure 11: The hydrophobic interactions found in the crystal structure of GAR300-Gp peptide in complex with mutant MDM2
interface. a) Two H-bounds formed (green dashed lines) between Phe19 and Thr18 residues of the peptide helix and the carbonyl oxygen of Gln72 residue in MDM2 cleft. b), c) Additional hydrophobic interactions (yellow dash-lines) between the peptide helix and MDM2 interface, the involved residues are shown as sticks.
4 Conclusion
Drug design and discovery has evolved in the recent decade to overcome challenging and fatal diseases including cancer. Computational and modeling of molecules to disrupt a specific PPI become a promising and feasible way to tackle human diseases. P53-MDM2 is the most explored PPI and is considered as a major drug target to treat cancers with WT-p53 by blocking this interaction and rescuing the biological function of WT-p53. The hydrophobic interface of MDM2 has became an attractive surface for designing small molecule compounds that showed successful results allowing these molecules to reach human clinical trials (Ortiz et al. 2016; Estrada-Ortiz et al. 2017; Ray-Coquard et al. 2012). However, small molecules cannot bind potently to all targeted interfaces due to their flat nature and inconsistency in conformation and amino acids composition (Jochim et al. 2010; Meireles et al. 2011; Verdine and Hilinski, 2012). All together
a) Gln72 Thr19 Phe19 Phe19 Trp23 Ile61 Gly58 b) His96 Leu26 Tyr22 His73 Val93 c)
expanding into new targets and utilizing novel chemistry strategies to broaden their functional diversity as well as to enhance their pharmaceutical properties. With stapling, peptides become stable in reinforced α-helix folds that resist proteolytic degradation and have a high cellular permeability. In addition, the digestion of stapled peptides is non-toxic and they have rapid clearance by renal filtration (Jenssen and Aspmo, 2008). This new class of inhibitors had huge success in targeting p53-MDM2 PPI as found in the complex crystal structures (PDB 3V3B, 4UMN, 5AFG, 4UE1, 4UD7 and 5XXK). Consequently, in our recent work we report three high-resolution novel structures of stapled peptides in complex with MDM2 (L33E). These peptides were designed based on WT-p53 sequence with a staple introduced at i,i+4 space position and were synthesized using the multi-component reaction (MCR) technique - the first to report the use of MCR for staple synthesis. Two peptides were found to form hydrophobic interactions between their staple and MDM2 surface “GAR300-Am and Gm” that could be linked to the meta position of the benzene ring of the staple of both peptides. This type of intimate staple-protein hydrophobic−hydrophobic interaction was also observed in the crystal structure of SAH-p53-8 and E1stapled peptides in complex with MDM2 (Baek et al. 2012; Lau and Wu, 2015), MCL-SAHBD in complex with MCL-1 (Stewart et al. 2010) and SP2 and SP2 in complex with estrogen receptor (Phillips et al. 2011). However, our structures disclosed unique features that are not found in any other reported MDM2 structures, in that the staple itself forming an interaction with MDM2 interface. Furthermore, our findings support the concept that stapling increase the binding toward the target intracellular protein as we found in the MST titration experiments of the stapled peptides with WT-MDM2; and the Kd values of 31, 2.4 and 3.14 nM for GAR300-Am, GAR300-Gp, and GAR300-Gm, respectively (relative to the WT-p53 peptide with Kd of 600 nM). Finally, stapled peptide-mediated disruption of intracellular PPIs is a recent advance and could be the basis for next-generation protein-based drugs that might target surfaces that cannot be addressed with traditional small molecules or biologics. There is still much to be learned, regarding stapled peptides development - including, peptide permeability and delivery, formulation and half-life. Efforts are underway to improve these parameters to increase peptide oral bioavailability and specificity in order to generate a unique class of potent molecules inhibiting PPIs.
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