University of Groningen Development of novel anticancer agents for protein targets Estrada Ortiz, Natalia

Development of novel anticancer agents for protein targets

Estrada Ortiz, Natalia

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CHAPTER

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OW TO DESIGN A SUCCESSFUL P

53-

MDM

2/

X

INHIBITOR

:

A THOROUGH OVERVIEW BASED ON

CRYSTAL STRUCTURES

Natalia Estrada-Ortiz, Constantinos G. Neochoritis and Alexander Dömling*

Department of Drug Design, University of Groningen Antonius Deusinglaan 1, 9700 AD Groningen, The Netherlands.

Abstract: A recent therapeutic strategy in oncology is based on blocking the protein-protein interaction MDM2/X-p53. Inhibiting the binding between wild-type p53 and its negative regulators MDM2 and/or MDMX has become an important target in oncology to restore the anti-tumor activity of p53, the so-called guardian of our genome. Interestingly, based on the multiple disclosed compound classes and structural analysis of small molecule-MDM2 adducts, the p53-MDM2 complex is perhaps the best studied and most targeted protein-protein interaction. Several classes of small molecules have been identified as potent, selective and efficient inhibitors and many co-crystal structures with the protein are available. In this report we are describing properties, preclinical and clinical studies of the small molecules and peptides classified in categories of scaffolds. A special focus is on crystal structures and the binding mode of the compounds including conserved waters.

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1. INTRODUCTION

Protein-protein interactions are of outmost importance and implicated in almost all biological processes. Proteins should not be considered to function as single, isolated entities but display their roles by interacting with other cellular components and the different interaction patterns of a protein is at least as important as the intrinsic biochemical activity of the protein itself. Therefore, the biological role of a protein is heavily based on the protein-protein interactions. Especially for diseases, the majority of cases ultimately relies on regulating the PPIs. Identifying and successfully targeting the PPIs by finding inhibitors or activators is the basis of the drug discovery.1

P53 is the principal regulator of the cell division and growth,2,3 _{being able to control genes implicated}

in cell cycle control, apoptosis, angiogenesis metabolism, senescence and autophagia.4 Mutations in this protein are present in about 50% of human cancers; altering the DNA binding domain ceasing p53’s activity as a transcription factor.5 _{The remaining tumors, p53 pathway is inactivated by}

up-regulation of p53 inhibitors, such as the mouse double minute proteins (MDM2 and MDMX, -also known as MDM4- HDM2 and HDMX in humans), or by down-regulation of p53 cooperators, such as ARF.6,7

The MDM2 gene was found to be upregulated in approximately 7% of tumors, with increased transcript levels and enhanced translation. Mutation of p53 and upregulation of MDM2 do not usually occur within the same tumor, indicating that MDM2 over-expression is an effective path for inactivation of p53 function in tumorgenesis.8 MDM2 functions as an inhibitor of the N-terminal trans-activation domain (TAD) of p53, and promotes p53 degradation through the ubiquitin-proteasome system (E3 ligase activity).9,10 _{On the other hand, MDMX can downregulate p53 via inhibition of the}

TAD domain, and it can upregulate MDM2.11

Moreover, it does not have E3 ligase activity, but its binding with MDM2 increases the rates of ubiquitinylation of p53 by MDM2.11,12 _{The C-terminal RING domains of both MDM2 and MDMX, is}

involved in dimerization; MDM RING domains can form homodimers, the heterodimers can be form by a reduced autoubiquitylation of MDM2 and increased p53 ubiquitylation.13 _{Consequently, MDM2 is}

stabilized by MDMX, keeping the levels of p53 low in healthy cells.13–15 _{The use of dual action}

MDM2/MDMX antagonists in cancer cells expressing wild type p53 should activate p53 more significantly than agents that only inhibit MDM2, resulting in more effective anti-tumor activity.16–18

The p53-MDM2 interaction is well druggable by small molecules based on a buried surface area of ~700 Å2, a well-structured deep and hydrophobic binding site of similar dimension than small molecules. For comparison the immune-oncology target protein-protein interaction PD1-PD1L has a buried surface area of ~2000 Å2, and is flat and featureless (figure 1). 19,20

The binding site of p53 in MDM2 is formed by 14 residues: Leu57, Met62, Tyr67, Gln72, Val75, Phe86, Phe91, Val93, His96, Ile99 and Tyr100. The cleft at the surface of MDMX is highly similar, 4 out of 14 residues are however different: MDM2_Leu54>MDMX_Met53, MDM2_Phe86>MDMX_Leu85, MDM2_His96> MDMX_Pro95

and MDM2Ile99>MDMXLeu98.18 MDM2 and MDMX have a deep hydrophobic pocket on which the p53 protein binds as an alpha helix. Three p53 amino acids are deeply buried in the MDM2 and MDMX clefts: Phe19, Trp23, and Leu26 and the interaction is mostly governed by hydrophobicity (PDB: 1YCR, figure 1).21,22

Figure 1. Two important protein-protein interaction targets: left p53 (green surface) with MDM2 (grey surface), PDB: 1YCR; below: footprint of p53 on MDM2 shown as blue surface. Right: PD1 (green surface) with PD1L (grey surface, PDB: 4ZQK); below: footprint of PD1 on PD1L shown as blue surface

Figure 2. p53 key amino acids Phe19, Trp23 and Leu26 bound to MDM2 (PDB: 1YCR). Red dotted lines indicate the polar contacts

Additionally, the Trp23 indole-NH forms a hydrogen bond with the backbone carbonyl of MDM2_{Leu54. In}

MDMX, Met53 and Tyr99 are bulged into the hydrophobic surface groove making it smaller and slightly different in shape (PDBs: 3DAB, 3DAC).21 The known three finger pharmacophore model for p53-MDM2 is now widely accepted to be responsible for the binding of small molecules and peptides to the MDM2.23–25 Recently an extended four finger model was proposed taking into account the intrinsically disordered MDM2 N-terminus which can be ordered by certain small molecules as shown by co-crystallization.26,27

Due to the importance of p53-MDM2/X protein-protein interaction, several reviews have been published in the last years.28–31 This review is giving a broad recent overview of inhibitors of the p53-MDM2/MDMX protein-protein interaction, mostly focusing on the available structural data. Additionally, some insights into the rational design and optimization of MDM2/X binder, their activities in vitro, in vivo and clinical trials are given. Moreover, a preliminary water analysis of the current crystal structures is presented.

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2. I

53-MDM2/MDMX

2.1. N

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The first class of highly potent, specific and orally active MDM2 inhibitors, was disclosed in 2004 by scientists from Hoffmann-La Roche,32 _{cis-diphenyl substituted imidazolines, known as Nutlins (1).}

Modifications and optimizations resulted in derivatives named Nutlin 1 (1a), Nutilin 2 (1b) and Nutlin 3a (1c) (figure 3). Nutlin compounds show inhibitory concentration values (IC50) of 260, 140 and 90

nM, respectively. These compounds displaced recombinant p53 fragment (corresponding to residues 1-312 of human p53) from its complex with MDM2 using surface plasmon resonance in a competition assay (SPR).32 Nutlin 2 complexed with MDM2 co-crystal structure (PDB: 1RV1, figure 4) led to the elucidation, for the first time of a non-peptide, small molecule structural information of the interaction.32 _{Superimposition of the co-crystal structures MDM2-Nutlin 2 and MDM2-p53 (PDB:}

1YCR), showed that the two bromo-substituted phenyl rings and the ethoxyl group of Nutlin 2 mimic the Trp23, Leu26 and Phe19, respectively, the key hydrophobic binding residues of the p53 peptide (figure 1).32 _{Nutlin 3a bound to MDM2 (PDB: 4J3E)}33 _{shows that both 4-chlorophenyl groups perfectly}

fill the Leu26 and Trp23 pockets, while the isopropoxy group reaches deep into the Phe19 pocket. It activates wild-type p53 and selectively kills cancer cells, with IC50 of 1-2 μM in the SJSA-1, HCT116 and

RKO cell lines (osteosarcoma, colorectal and colon carcinoma, respectively) and showed 10-fold selectivity compared with p53 mutated cell lines MDA-MB-435 and SW480 (melanoma and colorectal adenocarcinoma, respectively).32 Furthermore, in vivo studies demonstrate the capability of Nutlin 3a to reduce tumor growth by 90% in SJSA-1 in mice xenograft model.32

In 2008, compound 2 (RG7112) based on Nutlins (PDB: 4IPF),34 completed phase I clinical trials against advanced solid and soft tissue tumors and hematological malignancies.35,36 Four key modifications were made to enhance the binding activity to MDM2, cellular growth inhibition and improve pharmacokinetic properties: Two methyl groups were introduced in the imidazole ring to protect from metabolism, the isopropyl ether was replaced to ethyl ether to reduce the molecular weight maintaining the hydrophobic interactions, the methoxy group was changed by a tert-butyl moiety to decrease the metabolic liability and lastly, methyl sulphonyl group was added onto the piperidine ring to increase the binding affinity and improve the pharmacokinetics via reduced logD. After these modifications, compound 2, displayed enhanced binding activity towards MDM2 with a Kd 10.7 nM

and cellular growth inhibition 3-fold more potent compared with Nutlin 3a.33 _{The main drawback}

shown during the clinical trials in patients with liposarcoma, was thrombocytopenia, but the data obtained from biopsies in this preliminary trial suggested that the p53 pathway can be reactivated despite the presence of excess of MDM2, resulting in cytostatic and possibly apoptotic effects in tumor cells (figure 3).35,37

O N N S O O N N Cl Cl O O _N NH N N O O Cl Cl O 1c (Nutlin-3a) 2 (RG7112) Cl N N CO2H NH Cl Cl N N NH Cl N O N 3 (WK-23) 4 (WK-298) Cl N N NH Cl NH O N O 5 N _N F Cl O N N NH2 F Cl HN O 7 N _N Cl OH O Cl 6 N O NH O S N N Cl Cl S N N Cl Cl O N Cl O S N N Cl N NH O 8 9 10 N N N O O N N OH N N O O Br Br O N N O N N O O Cl Cl 1b (Nutlin-2) 1a (Nutlin-1)

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Figure 4. Crystal structure of Nutlin-2 bound to MDM2 (PDB: 1RV1) Nutlin-2 (green sticks) receptor (grey sticks) interactions are indicated by colored dotted lines: yellow; ŚLJĚƌŽƉŚŽďŝĐ͕ŽƌĂŶŐĞ͖ʋ-ʋ͕ďůƵĞ͖ŚLJĚƌŽŐĞŶďŽŶĚŝŶŐ͕ďůĂĐŬ͖ cation dipolar, chocolate; halogen, red; dipolar interactions. The receptor and compound are aligned with the p53 hot spot amino acids (magenta lines, PDB: 1YCR) (color code and the alignment maintained throughout the remaining figures). The 3-(4-ďƌŽŵŽƉŚĞŶLJůͿƵŶĚĞƌŐŽĞƐĂʋ-ʋƐƚĂĐŬŝŶŐƚŽ,ŝƐϵϲĂŶĚŝƐĞŵďĞĚĚĞĚŝŶŚLJĚƌŽƉŚŽďŝĐŝŶƚĞƌĂĐƚŝŽŶƐǁŝƚŚ Leu54; the 2-(4-bromophenyl) residue is making multiple hydrophobic contacts to Leu54, Gly58, Ile61 and Val93.

2.2. I

I

Tri-substituted imidazole compounds 3-5 with a 6-chloroindole moiety as tryptophan mimic were discovered by a virtual Trp23-anchor based pharmacophore screening approach and validated as MDM2 inhibitors (figure 3).38 _{The side chain of p53-Trp23 is embedded in a deep hydrophobic pocket}

formed by the MDM2 residues Leu57, Phe86 and Ile99 and it was noted early on that at the bottom of the indole-Trp23 unfilled hydrophobic space is left which could be filled by a suitable hydrophobe.39 Thus substitution of the indole in the 6-position by a methyl, alkynyl group or halogen atom can enhance the binding affinity by a factor up to 50. In addition, two other fragments were introduced to mimic leucine and phenylalanine residues, leading to 3 (WK23), co-crystalized with MDM2 (PDB: 3LBK) with a binding affinity 916 nM and 36 μM for MDM2 and MDMX, respectively. Its analogue 4 (WK298) with a binding affinity 109 nM and 11 μM for MDM2 and MDMX, respectively, comprises the first co-crystal structure of a small molecule bound to MDMX (PDB: 3LBJ).40 These crystal structures clearly reveal that 1,3,5-trisubstituted 5-indolo imidazoles compounds can bind both to MDM2 and MDMX and thus provide a starting point for the design of dual action MDM2/X antagonists. Compound 3 binds to MDM2 by filling its Trp23 subpocket with the 6-chloroindole group, the 4-phenyl group is located in the Phe19 and 1-(4-chlorobenzyl) group in the Leu26 pockets. A hydrogen bond is formed between the indole of 3 and the Leu54 carbonyl oxygen of MDM2 similar to the Trp23-Leu54 hydrogen bond found in the endogenous p53-MDM2 interaction (figures 3 and 5).40 _{The inhibitor 4}

binds to MDMX in a similar way, despite the different shapes of the p53 binding sites in MDM2 and MDMX. The His96-Tyr100 region has the most pronounced differences in the shape of the Leu26 pocket, but the position of 1-(4-chlorobenzyl) is not altered. Two hydrogen bonds with MDMX_{Met53 and} MDMX

His54 are formed, whereas the N,N-dimethylpropylamine part of 4 folds over MDMXGly57 and

MDMX_{Met61, forming additional hydrophobic protection of the binding cleft, where} MDMX_{Tyr99 closes}

the p53_{Leu26 subpocket.}40

Furthermore, the 3-imidazolyl indole inhibitor 5 (PDB: 4DIJ) was reported based on the central valine concept of Novartis by which a planar aromatic ring (imidazole) was placed in Van der Waals contact with the side chain of Val93, occupying a central position in the upper part of the MDM2 pocket and providing different type of substitutions.41 /Ŷ ƚŚŝƐ ƐƚƌƵĐƚƵƌĞ͕ ĂŶ ĂƌŽŵĂƚŝĐ ʋ-ʋ ƐƚĂĐŬŝŶŐ ŝŶƚĞƌĂĐƚŝŽŶ between MDM2 residue His96 and the benzylic chlorophenyl ring of the compound was observed.

Attempts to optimize compound 5 either by promoting the formation of hydrogen bond with His96 and/or improving the cellular activity, led to the tetra-substituted imidazoles 6 (PDB: 4OQ3) and 7. Modeling showed that the plane of the inhibitor core imidazole ring and that of its chlorophenyl ring are nearly perpendicular, with an angle of 80o (figure 3).41,42

Figure 5. Crystal structure of compound 3 (WK23) bound to MDM2 (PDB: 3LBK): A hydrogen bond between the indole N-H of 3 and the MDM2Leu54 carbonyl oxygen is depicted. The 1-(4-chlorobenzyl) group undergoes a T-ƐŚĂƉĞĚ ʋ-ʋ stacking to His96. The compound is making multiple hydrophobic interactions with Leu57, Gli58, Ile61, Met62, Val93, His96 Ile99 and Tyr100.

ɁŽǀĞů ĚŝŚLJĚƌŽŝŵŝĚĂnjŽƚŚŝĂnjŽůĞ ĚĞƌŝǀĂƚŝǀĞƐ ĐŽŵƉƌŝƐŝŶŐ ĂůƐŽ Ă ĐŽƌĞ ŝŵŝĚĂnjŽůĞ ŚĞƚĞƌŽĐLJĐůĞ ǁĞƌĞ developed as potent inhibitors of the p53-MDM2 interaction. As an initial lead, scientists from Daiichi-Sankyo reported compound 8.[22] _{Further optimization by using a methyl group onto the C-6 position}

to avoid oxidation, and by modifying the C-2 moiety of the additional proline motif, led to compound 9 (PDB: 3VZV) with an IC50 of 8.3 nM in a homogeneous time-resolved fluorescence assay (HTRF).43,44

The pyrrolidine moiety at the C-2 position induced another hydrophobic interaction site with MDM2 protein (Met50, Tyr67), as the co-crystal structure analysis revealed (figure 6). Moreover, solubility was improved by introducing an alkyl group into the pyrrolidine at the C-2 substituent and modifying the terminal substituent of the proline motif. Compound 10 (PDB: 3W69) with an IC50 of 59 nM (HTRF)

exhibited also good pharmacokinetic profile and significant antitumor efficacy via oral administration on mice xenograft model using MV4-11 cells bearing wild type p53 (figure 3).43,44

Figure 6. Crystal structure of compound 9 bound to MDM2 (PDB: 3VZV): The pyrrolidine moiety induced a new hydrophobic interaction site with Met50 and Tyr67. Multiple hydrophobic interactions are formed with Thr26, Leu54, Ile61, Met62, Val93, Ile99 and Tyr100.

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2.3. I

S

Using the pharmacophore search software ANCHOR.QUERY,45 _{a series of 6-chloro-indole derivatives}

were discovered, based on Ugi multicomponent reaction chemistry (figure 8).46

A novel class of compounds derived from an Ugi multi component reaction (Ugi four component-five centers, U-5C-4CR) showed potent binding towards MDM2 and MDMX, via the classical three finger pharmacophore model, mimicking the hot spot amino acids Phe19, Trp23 and Leu26. The most potent compounds are 11 (PDB: 3TJ2), 12 (PDB: 4MDQ) and 13 (PDB: 4MDN) with Ki of 0.4, 1.2 and 0.6 μM,

respectively in a fluorescence polarization assay (FP), which was verified by heteronuclear single quantum coherence (HSQC) experiment where 15N labeled MDM2 is used and thus the ligand-induced perturbations in NMR chemical shifts are observed.26,46 _{Compound 13 exhibited a novel fourth}

pharmacophore binding motif in MDM2, that is directly connected to the Leu26 sub-pocket forming a deep binding spot around the fourth pharmacophore point of the molecule. A hydrogen bond is formed again between the indole of 13 and the Leu54 carbonyl oxygen of MDM2 mimicking the Trp23-Leu54 interaction. Furthermore, a halogen bond between the Cl of the p-chlorobenzyl moiety and the carbonyl groups of Glu23 is possibly formed (figure 7).26,46,47

Researchers from Hoffmann-La Roche, identified a series of indolyl hydantoin compounds as potent, dual MDM2/MDMX inhibitors. The most representative compound of the series, 14 (RO-2443, PDB: 3VBG) showed an outstanding inhibitory activity against both MDM2 and MDMX with IC50 for the

binding to p53 of 33 nM and 41 nM respectively. Additionally, RO-2443 binds to the p53 pocket of MDMX and crystallographic and biochemical investigations suggested protein homodimerization and heterodimerization as mechanism of action (PDB: 3U15).48 Due to poor water solubility it was initially not possible to assess cellular activity; thus after supplementary optimization, compound 15 (RO-5963) was derived which inhibited the binding of p53 to MDM2 and MDMX and showed strong antiproliferative activity in cancer cell lines overexpressing MDMX (figure 8).48 These dimerizer molecules comprise a Michael acceptor system and thus the observed antiproliferative effects observed cannot be clearly assigned to one mode-of-action.

Figure 7. Crystal structure of compound 13 bound to MDM2 (PDB: 4MDN): A hydrogen bond is formed again between the indole NH of 13 and the carbonyl oxygen of Leu54. The compound is making hydrophobic interactions with Glu23, Met50, Leu54, Phe55, Gly58, Gln59 Ile61, Met62, Val93, Ile99 and Tyr100.

N H Cl HO2C N O H O HN Cl N H O NH HO Cl O HN O NH HO N H HN O HO Cl O N O O Cl N H 11 N Cl NH O NH Cl F NH O O NH₂ O 23 (RO-8994) N Cl NH O NH Cl F NH O O NH2 O 24 (RO-2468) NH O NH Cl F NH O O NH2 O S Cl 25 (RO-5353) Cl NH O NH N O 19 (MI-5) Cl NH O NH H N O F Cl OH OH 20 (MI-219) Cl NH O NH H N O Cl F OH 21 (MI-888) 12 13 Cl NH O NH H N O Cl F 22 (SAR405838) OH N HN O O F F HN Cl 14 (RO-2443) N HN O O F F HN Cl H N O OH OH 15 (RO-5963) N H H3CO N 16 (SP-141) NH NH O H N O Cl NH2 Cl F 17 18 (MI-63) Cl NH O NH H N O F Cl N O

Figure 8. Indoles and spirooxindoles derivatives as inhibitors of p53-MDM2/X interaction.

Recently, by performing high-throughput virtual screening, a new class of pyrido[b]indole derivatives were discovered, with the most active compound of this class compound 16 (SP-141), possessing a Ki

value of 28 nM for the binding with MDM2 in a FP based assay. Significant inhibition of breast cancer cell growth, and decreased growth of tumors in two different breast cancer xenograft models were observed (figure 7).49 However the binding mode of 16 is unclear and does not fit the 3-point pharmacophore model shown by the rest of the compound discussed here.

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Another privileged structure for the p53-MDM2 inhibition is the spirooxindole. Based on the insight that an oxindole group can mimic the Trp23 moiety, spirooxindole-containing natural products were identified and docked to MDM2. A series of compounds e.g. 17 (PDB: 4JVR), discovered via high-throughput screening assays based on spirooxindoles, were developed with favorable results in HTRF assays showing IC50 of 9.4 nM.50 Compound 18 (MI-63) was also developed having a Ki of 36 nM and 55

uM on MDM2 and MDMX respectively.51 _{Analyzing the crystal structure of a MI-63 analogue (PDB:}

3LBL), the compound binds to MDM2 also by nesting the chlorophenyl substructure of the 6-chlorooxindole into the Trp23 subpocket. The Leu26 subpocket is filled by the 2-fluoro-3-chlorophenyl ring which is located as in compounds 3 (WK23) and 4 (WK298) but its plane is rotated to allow the phenyl substituent atoms to fill the bottom of the MDM2 pocket. The neopentyl fragment fills the Phe19 pocket and causes a substantial induced-fit reshaping of the binding cleft, the Tyr67 side-chain is rotated to close the binding region and the whole Tyr67-His73 region acquires a different fold to allow the Tyr67 movement. The compound forms two hydrogen bonds with Leu54 and His96. The ethyl-morpholino part of the compound is not taking part in the binding and is not seen in the electron density (figure 7).40,50

Thorough optimization of the scaffold led to compounds with Ki values in the low μM and nM range

(compounds 19-21), giving promising results in vitro and in vivo assays with compounds 20 (MI-219) and 21 (MI-888) showing Ki values of 8.5 μM and 5 nM (HTRF), respectively.22,50 An analogue of

compounds 20 and 21 which has been advanced into phase I clinical trials, is compound 22 (MI773, SAR405838). This compound exhibits Ki of 0.88 nM (10-fold more potent than MI-219). As the

co-crystal structure reveals the inhibitor 22 mimics the three important amino acids, captures additional interactions that were not observed in the p53-MDM2 complex and induces refolding of the unstructured N-terminal region of MDM2 to achieve its high affinity. Furthermore, 22 activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell-cycle arrest and/or apoptosis (figure 7).52

Based on MI-series of compounds, Hoffman la Roche scientists developed modified spirooxindole small molecules using an additional phenyl moiety to fit into the Phe19 pocket and an isopropyl group to mimic Leu26 (23, RO-8994) with an IC50 of 5 nM (HTRF).53,54 As expected, the

6-chloro-1,3-dihydro-2H-pyrrolo[3,2-c]pyridin-2-one moiety in compound 23 is buried into the Trp23 pocket, and its NH moiety forms a hydrogen bond with a backbone carbonyl of MDM2 for enhanced binding affinity. Since the interaction in the Trp23 pocket appears to be the most critical, further exploration of bioisosteric replacements on the phenyl moiety of 6-chlorooxindole, while preserving other important architectural features in RO8994 for optimal binding and pharmacological properties led to 24 (RO-2468) and 25 (RO-5353, PDB: 4LWV) with an IC50 of 6 and 7 nM (HTRF) respectively. In the latter, the

2-chlorothienyl[3,2-b]pyrrol-5-one group is buried into the Trp23 pocket, whereas the 3-chloro-2-fluorophenyl group occupies the Leu26 pocket (figure 9).53 _{Examples 23-25 nicely show that}

bioisosterc replacement of the abundant p-halogen substituted phenyl groups in the Trp23 pocket by heteroraromatic rings is possible and can be potentially used to improve the overall very hydrophobic properties of MDM2 antagonists.

Figure 9. Crystal structure of compound 25 (RO-5353) bound to MDM2 (PDB: 4LWV): Hydrogen bonds are observed both between the N-H group of the pyrrole and Leu54 and between the carbonyl of the amide and His96. Additionally ƚǁŽʋ-ʋƐƚĂĐŬŝŶŐŝŶƚĞƌĂĐƚŝŽŶƐĂƌĞĨŽƌŵĞĚďĞƚǁĞĞŶ,ŝƐϳϯǁŝƚŚƚŚĞϮ-methoxy-4-carbamoyl amide aromatic ring, and His96 with the 2-fluoro-3-chloro benzene ring. Hydrophobic interactions are observed with Leu54, Leu57, Ile61, Met62, Phe86, Val93, Ile99 and Tyr100.

2.4. P

After the discovery of compounds 2 (RG7112) and 23 (MI-219), novel pyrrolidine derivatives were also developed as p53-MDM2/X inhibitors by Roche scientists. The prototype compounds 26 and 27 (PDBs: 4JRG and 4JSC) showed good potency (IC50 = 196 nM and 74 nM respectively) in the HTRF assay.54 The

4-chlorophenyl ring of compound 26 is buried in the Trp23 pocket, the 3-chloro-phenyl occupies the Leu26 pocket and the neopentyl binds to the Phe19 pocket (figure 10).

Figure 10. Crystal structure of compound 26 ďŽƵŶĚ ƚŽ DDϮ ;W͗ ϰ:Z'Ϳ͗  ʋ-ʋ ƐƚĂĐŬŝŶŐŝŶƚĞƌĂĐƚŝŽŶ ǁŝƚŚ ,ŝƐϵϲ ŝƐ depicted. Additionally the pyrrolidine carbonyl forms a hydrogen bond with NH of His96. Several hydrophobic interactions are observed with Leu54, Leu57, Ile61, Met62, Phe86, Phe91, Val93, Lys94, Ile99 and Tyr100.

Further optimization led to the development of the compound 28 with an IC50 of 6 nM (RG7388),

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expressing wild-type p53 and tumor growth inhibition or regression of osteosarcoma xenografts in nude mice. RG7388 is undergoing clinical investigation in solid and hematological tumors (figure 11).54

NH O HN N F Cl F Cl 28 (RG7388) NH O NH HO HO N F Cl F Cl O _O OH 27 NH O NH HO HO N Cl Cl 26

Figure 11. Pyrrolidine derivatives screened for the suppression on p53-MDM2.

2.5. I

,

In 2012 Novartis scientists described substituted isoquinolines and piperidinones as inhibitors of MDM2 and MDMX with the most potent compound 29 having IC50 values of 0.8 and 2.1 μM,

respectively for both proteins in TR-FRET assay.55 _{Two additional patents reported the}

cyclohexylisoquinoline compound 30 and hydroxyl isoquinoline 31 which are also capable of inhibiting the interaction between p53-MDM2/X with IC50 ŽĨϬ͘ϴϵϰʅɀ;DDϮͿ͕ϰϰ͘Ϯϱʅɀ;DDyͿĂŶĚϬ͘ϰϯϮ

ʅɀ ;DDϮͿ ƌĞƐƉĞĐƚŝǀĞůLJ ;ĨŝŐƵƌĞ ϭϮͿ͘56,57 _{Recently, Novartis described a new series of}

dihydro-isoquinolinone derivatives derived from a virtual screening exercise using 2D and 3D presentations of ~50.000 compounds from the Novartis compound collection and compound 32 was identified with an IC50 ǀĂůƵĞŽĨϬ͘ϱϰʅDŝŶdZ-FRET biochemical assay. The binding mode of compound 32 in the MDM2

pocket obtained by docking, adjusts to the central valine concept.41 _{Further optimization yielded}

compound 33 (PDB: 4ZYC) with an IC50 ǀĂůƵĞ ŽĨ Ϭ͘ϯϴʅD ŝŶ dZ-FRET assay. Remarkably, the bicyclic

core makes hydrophobic contacts with residues Ile54, Phe55 and Gly58 of MDM2 instead of the proposed interaction with Val93.58 _{The development of this class of compounds led to compound 34}

(NVP-CGM097, PDB: 4ZYI) with IC50 = 1.7 nM with MDM2 and strong antiproliferative effect in the

HCT116 p53WT cell line compared to an isogenic HCT116 knockout for the expression of p53, showing 35-fold selectivity. Additionally, it was determined a 58-fold selectivity between SJSA-1 cell line and p53-null osteosarcoma SAOS-2 cell line.59 Presently, NVP-CGM097 is going through phase 1 clinical trials (figure 12).59,60

N O OMe O N N N O N O OMe Oi_Pr Cl OH N O OMe Oi_Pr Cl MeO 29 30 O OH OMe NH O N O Cl F Cl S O O 41 (AM-7209) O OH N O O Cl Cl S O O 42 (AM-8735) N O O O MeO N O O O Cl O N HN N N N O O O Cl N N N O 34 (NVP-CGM097) 31 32 33 O OH N O Cl Cl O OH N O Cl Cl O O O OH N O OH Cl Cl 35 36 37 O OH N O OH Cl Cl O OH N O S Cl Cl O O 38 (AM-8553) 39 (AM-232) O OH S N N O Cl F Cl S O O 40 (AM-6771) Cl

Figure 12. Various isoquinolines, piperidinones and morpholinones as potent inhibitors.

Scientists from Amgen described the structure-based design of new piperidinone compounds, based on yet another scaffold class.61 In compound 35, with an IC50 of 34 nM (PDB: 2LZG), the p-chloro

substituted phenyl ring occupies the Trp23 pocket, the m-chloro substituted phenyl ring sits in the Leu26 pocket and the cyclopropyl group fills the Phe19 pocket (figure 13). The carboxylic acid moiety forms a hydrogen bond with NH of His96 (figure 13).61,62

A chiral tert-butyl 2-butanoate replaced the cyclopropyl group in compound 35 and led to piperidinone 36, 8-times more potent than its predecessor towards MDM2 (PDB: 4ERE).61 _Further

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9

optimization led to the compound 38 (AM-8553, PDB: 4ERF), with an IC50 of 1.1 nM towards MDM2

(figure 12), showing satisfactory tumor regression in mice SJSA-1 tumor xenograft model, demonstrating its strong antitumor activity.63 _{Supplementary development of AM-8553, with the}

introduction of the sulfonamide moiety in order to search interactions between glycine in a shallow, underutilized cleft of MDM2 surface, yielded to compound 39 (AM-232, PDB: 4OAS) with Ki = 0.045

nM, IC50 = 9.4, 11.3 and 23.8 nM in SJSA-1, HCT116 and ACHN (renal adenocarcinoma derived cell

line), respectively.64,65 Additional evaluations in seven p53 mutant cell lines, showed no significant effect on cell growth and in studies of SJSA-1, HCT116, A375 (malignant melanoma) and NCI-H460 (large cell lung cancer) xenograft models it were observed significant tumor regression.64 _The

evaluation of AM-232 in preclinical trials, in rats, monkeys and dogs, showed differences in the pharmacokinetics with a good correlation between in vivo and in vitro studies. This methodology led to a prediction of low human plasma clearance and long half-life in human for compound 39.66

Currently, the compound is undergoing clinical trials (figure 12). 64–67

Figure 13. Crystal structure of compound 35 bound to MDM2 (PDB: 2LZG): The 3- chlorophenyl ring has a ʋ-ʋ stacking interaction with His96; additionally the carboxylic acid moiety forms a hydrogen bond with NH of His96. Numerous hydrophobic interactions with Val14, Leu54, Gly58, Ile61, Tyr67 and Val93 are shown.

In 2014, additional efforts by Amgen workers to enhance the biochemical and cellular potency, using pyridine or thiazole as isosteres of the carboxylic acid moiety of AM-232 led to potent piperidinone inhibitors, among them compound 40 (AM-6761, PDB: 4ODE), a thiazolyl-containing inhibitor with a HTRF IC50 = 0.1 nM, SJSA-1 IC50 = 16 nM, antitumor activity in the SJSA-1 osteosarcoma xenograft

model with an ED50 of 11 mg/kg and promising pharmacokinetic properties.68 Furthermore, an

additional modification of AMG-232, replacing the carboxylic acid with a 4-amidobenzoic acid, led to compound 41 (AM-7209, PDB: 4WT2), with a Kd value of 38 pM, SJSA-1, IC50 = 1.6 nM, in vivo

antitumor activity in SJSA-1 and HCT116 xenograft model and outstanding pharmacokinetic properties.69 Using the knowledge gained from the piperidinone series, changing to a morpholinone core had a significant impact on potency and metabolic stability. Morpholinone inhibitors are 5- to 10-fold less potent than their piperidinone counterparts, however they are more stable in hepatocytes, a feature can compensate the reduction in potency. Compound 42 (AM-8735, PDB: 4OBA), the most representative compound of this series showed an IC50 = 25 nM in SJSA-1 cells, remarkable

pharmacokinetic properties and in vivo efficacy in a SJSA-1 osteosarcoma xenograft model (ED50 = 41

2.6. B

In 2005 Johnson & Johnson reported a class of benzodiazepine compounds as MDM2 inhibitors (figure 14).71,72 _{With an initial high-throughput screening, two lead compounds were identified with a binding}

affinity with MDM2 between 15 and 30 μM, after further optimization of the substituents on the three phenyl rings resulted in compound 43 with IC50 сϬ͘ϮϮʅɀĂŶĚKd = 80 nM.73

N H N I O CO2H O Cl Cl 43 N N I Cl Cl NH₂ O O O O N H N O CO2H S Cl Cl Leu-Thr-Phe-HH₂N O O Glu-Tyr-HN O Ala-Gln NH H N O O Ser-Ala-Ala-NH2 46 (ATSP-7041) 44 45

Figure 14. Benzodiazepines and a staple peptide showing potency as MDM2/X inhibitors

Co-crystal structure of MDM2 and 43 S,S-isomer at a resolution of 2.7 Å (PDB: 1T4E) showed that that the binding resembles the three key p53 residues for interaction with MDM2.73 The Phe19 pocket was filled by the iodobenzene ring, the Trp23 pocket was occupied by the p-chlorophenyl and the Leu26 pocket was filled with other p-ĐŚůŽƌŽƉŚĞŶLJů Ăƚ ƚŚĞ ɲ-position corresponding to the carboxylic acid group (figure 15). The iodo substituent of the benzodiazepinedione scaffold forms a strong halogen bonding with the backbone carbonyl-O of Gln72. As expected a study reduction of affinity is observed be switching to lower halogens.71

Additional optimization resulted in compound 44 by reducing epimerization issues comparing with the ƐƚĞƌĞŽŐĞŶŝĐ ĐĞŶƚĞƌĂƚ ƚŚĞɲ-carbonyl position of 43 and 44 introduced a hydrogen bond with Val93 with IC50 ĨŽƌƚŚĞďŝŶĚŝŶŐǁŝƚŚDDϮŽĨϬ͘ϰϵʅɀĂŶĚϯϵϰŶD͕ƌĞƐƉĞĐƚŝǀĞůLJ͘74 Any of this class of MDM2

inhibitors progressed into clinical development due to their unsatisfactory antitumor activity in human cancer cells, low cell permeability, rapid plasma clearance and low bioavailability shown in vitro and in animal models.28,29,74 _{In 2012, a 2-thiobenzodiazepine derivative (compound 45) was described in a}

patent as a MDM2 inhibitor, with an IC50 of 3.18 μM/mL in Saos-2 p53 null and 6.54 μM/mL in U-2-os

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9

Figure 15. Crystal structure of compound 43 bound to MDM2 (PDB: 1T4E): A halogen bond between the iodine and Gln72 is observed. Hydrogen bond between Ser17 and the carboxylɿc acid moiety and a ʋ-ʋ stacking interaction with His96 and the p-chlorophenyl. Furthermore, various hydrophobic interactions with Leu54, Gly58, Ile61, Tyr67, Phe91, Val93, Ile99 and Tyr100 are shown.

2.7. P

Peptides and derivatives also can be designed to become potent p53-MDM2/X inhibitors. In 2007, a 12-residue peptide (pDI) was identified using phage display that selects for maximal inhibitory activity against MDM2 and MDMX, with IC50 = 0.01 and 0.1 μM for the binding in ELISA assay for MDM2 and

MDMX, respectively.76 The co-crystal structures of the pDI with MDMX (PDB: 3JZO) and a single mutant derivative (pDI6W, PDBs: 3JZP, 3JZR) bound to human MDMX and MDM2, served as template to design of 11 diverse pDI-derivative peptides that were tested for inhibitory potential. The best derivative (pDIQ, PDB: 3JZQ) exhibited a 5-fold increase in potency over the parental peptide with IC50

for the binding with MDM2 and MDMX of 8 and 110 nM, respectively.77

While peptidic inhibitors based on the modified p53 sequence offer very high affinity toward MDM2, they suffer from low cell permeability and are proteolytically unstable. A successful attempt to overcome these problems has been made by designing cyclic peptides that are closed by an all-hydrocarbon “staple”. The staple stabilizes the helical structure of the peptide, a feature which likely contributes to the enhanced affinity of the peptide for MDM2 relative to the wild-type peptide. The peptide SAH-p53-86 (PDB: 3V3B), which interestingly also targets MDMX, is the most effective stapled-peptide MDM2 inhibitor using its Phe19, Trp23, and Leu26 to fill the binding site in a manner similar to the native p53 peptide. The Trp23 indole ring is bound to Leu54 by a hydrogen bond. The aliphatic staple protects this hydrogen bond and forms an extended hydrophobic network with Leu54, Phe55, Gly58, and Met62 of MDM2 (figure 16).78

Figure 16. Crystal structure of the peptide SAH-p53-86 bound to MDM2 (PDB: 3V3B): The staple is indicated in pink. The hydrogen bonds are the same present in the p53-MDM2 interaction, the NH in SAH-p53-86

Trp23 and the carbonyl in

MDM2

Leu54, and between the NH in SAH-p53-86

Phe19 and MDM2

Gln72. Additionally, several hydrophobic interactions are depicted.

The PMI peptide (PDB: 3EQS bound to MDM2 and PDB: 3EQY bound to MDMX) a 12-mer peptide selected from a phage displayed peptide library, is highly soluble in aqueous solutions and has better affinity (compared to pDI) for MDM2 and MDMX with Kd values of 3.3 and 8.9 nM, respectively.79

Aided by mirror image phage display and native chemical ligation, several proteolysis-resistant duodecimal D-peptide antagonists of MDM2, termed D_{PMI-ɲ͕ɴ͕ɶ͕ɷǁĞƌĞĚŝƐĐŽǀĞƌĞĚ͘}80 _{The prototypic}

D-peptide inhibitor DPMI-ɲďŝŶĚƐƚŽDDϮ;W͗ϯ>E:ͿǁŝƚŚĂŶĂĨĨŝŶŝƚLJŽĨϮϮϬŶD͕ƌĞĂĐƚŝǀĂƚĞƐƚŚĞƉϱϯ pathway in tumor cells in vitro and inhibits tumor growth in vivo. Furthermore, the design of a superactive D-peptide antagonist of MDM2 was reported, named D_{PMI-ɷ;W͗ϯdWyͿ͕ǁŝƚŚĂďŝŶĚŝŶŐ}

affinity for MDM2 improved over DPMI-ɲďLJϯŽƌĚĞƌƐŽĨŵĂŐŶŝƚƵĚĞ;<d = 220 pM).81

In 2013, the stapled peptide 46 (ATSP-7041, PDB: 4N5T, co-crystallized with MDMX) was synthesized which activates the p53 pathway in tumors in vitro and in vivo, with Ki of 0.9 and 6.8 nM for MDM2

and MDMX, respectively. This stapled peptide has shown improved bioavailability compared to previously described peptides and induces p53-dependent apoptosis and inhibits cell proliferation in multiple MDM2 and MDMX overexpressing tumors in cell based models. Interestingly, beyond the well-known triad of Phe19, Trp2, and Leu26, ATSP-7041 demonstrated additional interactions between Tyr22 and the staple moiety itself with the MDMX protein. The Tyr22 interacts with MDMX binding pocket through Van der Waals contact with Gln66, Arg67, Gln68, His69, Val89, and Lys90 as well as through water-mediated hydrogen bonds with Nɸ of the Lys90 side-ĐŚĂŝŶĂŶĚEɷϭŽĨƚŚĞ,ŝƐϲϴ side-chain. An extensive binding pocket for the staple exists on the MDMX protein, and a number of van der Waals contacts (to Lys47, Met50, His51, Gly54, Gln55, and Met58) were also observed (figure 14).82

The MDM2-binding stapled peptide, M06, showed high affinity for the binding with mutant MDM2 (Nutlin resistant). The M06 stapled peptide forms interactions very similar to those made by the SAH-8 peptide, including the reorientation of the Leu26 side chain that appears to be associated with increased helicity (PDB: 4UMN). Apart from attenuating the binding of Nutlin, the M62A mutation in MDM2 also causes a significant change in the conformation of the aliphatic staple. In the SAH-8 structure the hydrocarbon chain packs predominantly against Leu54, Phe55, Gly58 and Met62. The absence of the methionine side chain in MDM2-Met62A causes the conformation of the staple to

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9

change as it packs more closely against Gly58. The plasticity of the new designed stapled peptide enables it to respond to the M62A mutation by making the compensatory contacts.83

3. C

Recently, plenty novel small molecules with promising activity have been published as MDM2 inhibitors, including a new synthetic approach to obtain stapled peptides,84 _{chlorofusin inspired class}

of analogs,85 _{fluorescent triazolylpurines,}86 _{sulfamide and triazole benzodiazepines,}87

oxazoloisoindolinones,88 dispiro-indolinones,89 a camptothecin analogue (FL118),90 and an inauhzin analogue,91 _{among others. Very recently, a 2,3ʚ-bis(1ȹ-indole) scaffold was published by our group}

showing additional hydrophobic interactions with the p53_{Val93 as indicated by 2D NMR and modelling}

studies.92 These compounds possess often modest affinities to MDM2 or MDMX in the micromolar range. All potent MDM2 antagonistic scaffolds currently undergoing early clinical trials have been structurally characterized and SBDD played an important role in their optimization. Thorough analysis of the available structure and SAR can provide some guiding principles for the design of novel and potent p53-MDM2 antagonists. The endogenous interaction of p53 with MDM2 and MDMX reveals a clear 3-point pharmacophore model formed by the side chains of p53 Phe19-Trp23-Leu26, which competitive inhibitors have to mimic. Multiple scaffolds have been designed capable of doing so and have been discussed in the previous sections. All structurally characterized MDM2 binder have a T-shaped topology where the ends of the T comprise the three moieties addressing the pharmacophore points. The central scaffold is mostly a heterocyclic ring, annulated rings or in some cases acyclic linear. Amongst the three buried hydrophobic amino acids side chains Trp23 contributes most to the interaction energy and thus must be closely mimicked in terms of hydrophobicity and shape. This has been accomplished by phenyl groups, indoles, oxindoles or 2-oxo benzimidazoles. The choice of the right anchor residue mimicking Trp23 might be important in terms of compounds selectivity. A considerable increase in binding affinity can be reached by the suitable introduction of a hydrophobic halogen at the bottom of the Trp23 binding pocket. A per-p-halophenyl substituted inhibitor scaffold might potentially bind into other similar ɲ-helix mediated protein-protein interactions, whereas an anchor residue closely mimicking the Trp23-Leu54 hydrogen bond might be more selective and able to differentiate between similar PPI binding interfaces. Additional interactions to receptor amino acids sees in several cocrystal structures involve a hydrogen bonding or ʋ- ʋ interaction to His96, hydrogen bonding to Ser17, halogen bonding or hydrogen bonding to backbone or sidechain Glu72, respectively, and dipolar interaction with His73. In general the MDM2 crystal structures show a high degree of crystallographic water presence but a water on top of the indole ring of p53Trp23 seems to be highly conserved in many structures. In 17 out of 25 MDM2 structures (~70%) a highly conserved water molecule can be observed (figure 17). This water is involved in a tight network of interactions between a polar inhibitor substituent on top of the central scaffold and the main chain Phe55 carbonyl-O in the MDM2 receptor. The inclusion of this water network could be an important aspect to consider into future design of MDM2 inhibitors.

Dual activity both in MDM2 and MDMX is essential for further substantial development. Current small molecules do however not address the duality nor are potent MDMX inhibitors known. In contrast peptidic inhibitors can be designed with dual potency for MDM2 and MDMX. In addition the intrinsically disordered protein states of MDM2 should be taken into account and the resulting 4th pharmacophore point, for discovery of novel scaffolds as shown in previous studies.26,27

Drug combination, and cyclotherapy had been studied as supplementary opportunities to treat cancer, exerting synergistic effects or protect selectively normal tissues.29 Representatives are the evaluation of Nutlin with actinomycin D, gemcitabine, vincristine, roscovitine and doxorubicin in cell based models.93–98 _{Another highly interesting and complementary p53 reactivation approach not}

The research efforts during the last decade led to several compounds currently in clinical trials for the treatment of different types of cancer and also in combination with other classes of chemotherapeutics (table 1). The available structural information and knowledge gather during the last years helped to design better compounds with greater affinity towards MDM2, but not in the same degree for MDMX, improved pharmacokinetic profile, oral bioavailability and selectivity. During the studies with animals as well as in clinical trials is imperative the determination of proper doses, schedules and possible combination therapies to circumvent possible acquired resistance to MDM2 inhibitors and side effects.

Figure 17. Highly conserved surface water in ligand-MDM2 structures. Above: Superimposition of the co-crystal structures including the waters, main cluster (shown in cyan) located in the groove formed by Phe55, Tyr56, Lys57, Gly58 and Gln59, Below: Examples of the interaction of the small molecules, the water and Phe55 of MDM2. First row: Compounds 1c (Nutlin 3, PDB: 4J3E) and 3 (WK23, PDB: 3LBK). Second row: Compounds 11 (PDB: 3TJ2) and 40 (AM-6761, PDB: 4ODE). Third row: Compounds 34 (NVP-CGM097, PDB: 4ZYI) and 43 (PDB: 1T4E).

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Table 1. MDM2/X inhibitors in clinical trials

Drug Condition Phase Sponsor Ref

RG7112 (RO5045337) Solid Tumors, advanced solid tumors, Leukemia, hem,atological neoplasms, liposarcomas

1 (completed) Hoffmann-La Roche 35–37,101

RG7112

(RO5045337) with Doxorubicin

Soft Tissue Sarcoma 1 (completed) Hoffmann-La Roche 102

RG7112

(RO5045337) with Cytarabine

Acute Myelogenous

Leukemia 1 (completed) Hoffmann-La Roche

103 RG7775 (RO6839921) Advanced Cancers, Including Acute Myeloid Leukemia

1 (Recruiting) Hoffmann-La Roche 104

RO5503781

Advanced

Malignancies, Except Leukemia

1 (Completed) Hoffmann-La Roche 105

RO5503781 with

Posaconazole Solid Tumors 1 (Completed) Hoffmann-La Roche

106

RO5503781 with Cytarabine

Acute Myelogenous

Leukemia 1 (Recruiting) Hoffmann-La Roche

107

RG7388 (Alone and with Pegasys)

Polycythemia Vera and

Thrombocythemia

1 (Recruiting) Hoffmann-La Roche 108

MK-8242 Advanced Solid

Tumors 1 (Completed)

Merck Sharp & Dohme Corp.

109

MK-8242 -Cytarabine Acute Myelogenous

Leukemia 1 (Completed)

Merck Sharp & Dohme Corp.

Drug Condition Phase Sponsor Ref DS-3032b Hematological Malignancies, advanced tumors or lymphomas

1 (Recruiting) Daiichi Sankyo Inc. 111

HDM201 Advanced Tumors

TP53wt 1 (Recruiting) Novartis

112

HDM201 with LEE011 Liposarcoma 1 (Recruiting) Novartis 113

CGM097 Solid Tumor With p53

Wild Type Status 1 (Recruiting) Novartis

60,114

SAR405838 (MI-888

analogue) Neoplasm Malignant 1 (Active) Sanofi

115

SAR405838 (MI-888

analogue) with pimasertib Neoplasm Malignant 1 (Recruiting) Sanofi

116 AMG232 Advanced Solid Tumors or Multiple Myeloma 1 (Recruiting) Amgen 67 AMG232 Metastatic Melanoma 1b/2a (Recruiting) Amgen 117

AMG232 with Trametinib Acute Myeloid Leukemia

1b (Recruiting)

3

4

5

6

7

8

9

4. R

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