Restoration of p53 activity by inhibition of the p53-MDM2 interaction has been considered an attractive approach for cancer treatment. However, the hydrophobic protein-protein interaction surface represents a significant challenge for the development of small-molecule inhibitors with desirable pharmacological profiles. RG7112 was the first small-molecule p53-MDM2 inhibitor in clinical development. Here, we report the discovery and characterization of a second generation clinical MDM2 inhibitor, RG7388, with superior potency and selectivity.

The p53 tumor suppressor is a potent transcription factor that plays a key role in the regulation of cellular responses to stress. It is controlled by its negative regulator MDM2, which binds directly to p53 and inhibits its transcriptional activity. MDM2 also targets p53 for degradation by the proteasome. Many tumors produce high levels of MDM2, thereby impairing p53 function. Restoration of p53 activity by inhibiting the p53-MDM2 interaction may represent a novel approach to cancer treatment. RG7112 (2g) is the first clinical small-molecule MDM2 inhibitor designed to occupy the p53-binding pocket of MDM2. In cancer cells expressing wild-type p53, RG7112 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest, apoptosis, and inhibition or regression of human tumor xenografts. KEYWORDS: MDM2, p53, RG7112, protein−protein interaction, cancer p53 is a potent tumor suppressor that activates the transcription of a subset of genes controlling cell-cycle progression and apoptosis.1−3 Dysregulation of the p53 pathway, including mutation or deletion of the p53 gene and changes in downstream signaling molecules, is the most frequent alteration in human cancers.4 MDM2 is a negative regulator of p53 that binds the transactivation domain of p53 and inhibits its ability to activate transcription.5−8 MDM2 is also an E3 ubiquitin ligase that targets p53 for proteosomal degradation.9 In a variety of solid tumors and hematologic malignancies, MDM2 overexpression is one of the mechanisms by which the wildtype p53 function is impaired.10 Given the central role of MDM2 in regulating p53 activity and stability, developing small-molecule inhibitors of MDM2 could offer a novel approach to treating cancers. 11,12The crystal structure of a p53-derived peptide bound to the p53 binding domain of MDM2 revealed the existence of a deep hydrophobic clef on the surface of the MDM2 molecule. 13Three amino acid residues from the p53 peptide (Phe19, Trp23, and Leu26) play critical roles in the binding between the two proteins by projecting hydrophobic side-chains deep into the cavity of the MDM2 molecule. These structural features of the p53-MDM2 complex suggested the likelihood of identifying small-molecule inhibitors that can successfully block the interaction between the two proteins. Compounds with the ability to inhibit the binding between p53 and MDM2 have been reported. 14−17 We previously reported the discovery of a series of 4,5-dihydroimidazolines called Nutlins. These compounds, exemplified by compound 1 (Figure 1), were discovered through screening and subsequent medicinal chemistry optimization. 18 Compound 1, also known as Nutlin-3a, has become a tool of choice to study p53 biology and therapeutic applications.19 Although these early lead compounds have shown good cellular activity and provided the mechanistic proof-of-concept for inhibiting p53-MDM2 interaction for cancer therapy, their pharmacological properties were suboptimal for clinical development. Here, we describe

Molecular features of ligand binding to MHC class II HLA-DR molecules have been elucidated through a combination of peptide structure-activity studies and structure-based drug design, resulting in analogues with nanomolar affinity in binding assays. Stabilization of lead compounds against cathepsin B cleavage by N-methylation of noncritical backbone NH groups or by dipeptide mimetic substitutions has generated analogues that compete effectively against protein antigens in cellular assays, resulting in inhibition of T-cell proliferation. Crystal structures of four ternary complexes of different peptide mimetics with the rheumatoid arthritis-linked MHC DRB10401 and the bacterial superantigen SEB have been obtained. Peptide-sugar hybrids have also been identified using a structure-based design approach in which the sugar residue replaces a dipeptide. These studies illustrate the complementary roles played by phage display library methods, peptide analogue SAR, peptide mimetics substitutions, and structure-based drug design in the discovery of inhibitors of antigen presentation by MHC class II HLA-DR molecules.

BioMAX is the first macromolecular crystallography beamline at the MAX IV Laboratory 3 GeV storage ring, which is the first operational multi-bend achromat storage ring. Due to the low-emittance storage ring, BioMAX has a parallel, high-intensity X-ray beam, even when focused down to 20 µm × 5 µm using the bendable focusing mirrors. The beam is tunable in the energy range 5–25 keV using the in-vacuum undulator and the horizontally deflecting double-crystal monochromator. BioMAX is equipped with an MD3 diffractometer, an ISARA high-capacity sample changer and an EIGER 16M hybrid pixel detector. Data collection at BioMAX is controlled using the newly developed MXCuBE3 graphical user interface, and sample tracking is handled by ISPyB. The computing infrastructure includes data storage and processing both at MAX IV and the Lund University supercomputing center LUNARC. With state-of-the-art instrumentation, a high degree of automation, a user-friendly control system interface and remote operation, BioMAX provides an excellent facility for most macromolecular crystallography experiments. Serial crystallography using either a high-viscosity extruder injector or the MD3 as a fixed-target scanner is already implemented. The serial crystallography activities at MAX IV Laboratory will be further developed at the microfocus beamline MicroMAX, when it comes into operation in 2022. MicroMAX will have a 1 µm × 1 µm beam focus and a flux up to 1015 photons s−1 with main applications in serial crystallography, room-temperature structure determinations and time-resolved experiments.

The development of small-molecule MDM2 inhibitors to restore dysfunctional p53 activities represents a novel approach for cancer treatment. In a previous communication, the efforts leading to the identification of a non-imidazoline MDM2 inhibitor, RG7388, was disclosed and revealed the desirable in vitro and in vivo pharmacological properties that this class of pyrrolidinebased inhibitors possesses. Given this richness and the critical need for a wide variety of chemical structures to ensure success in the clinic, research was expanded to evaluate additional derivatives. Here we report two new potent, selective, and orally active p53-MDM2 antagonists, RO5353 and RO2468, as follow-ups with promising potential for clinical development.KEYWORDS: MDM2, p53, wild-type, small molecule, apoptosis, cancer T umor suppressor p53 is a potent transcription factor that is activated in response to cellular stress and regulates downstream genes controlling cell cycle arrest and apoptosis. 1−4 Dysfunction of the p53 pathway is the most frequent alteration observed in human cancers. 5 MDM2 is the primary negative regulator of p53 through binding to its transactivation domain and promoting proteosomal degradation. 6−8 In tumor cells with wild-type p53 (∼50%), reactivation of the p53 pathway by inhibition of MDM2 with small molecules has been considered as potentially an attractive novel therapeutic approach for cancer treatment. 9,10 Currently, several smallmolecule MDM2 inhibitors including RG7112 and RG7388 (Figure 1) are undergoing clinical evaluations. 11−14 To maximize the chance of success in the clinic and derisk any potential idiopathic toxicity associated with specific chemotypes, continued research efforts are required to expand chemodiversity and identify potent and selective MDM2 antagonists with desirable in vitro ADMET and in vivo pharmacokinetic properties. Here we report the discovery of RO5353 and RO2468, two new highly potent and selective MDM2 inhibitors with potential for clinical development.Our exploration initially led to the identification of a potent and selective MDM2 inhibitor RO8994 (Figure 1), which was found to be highly efficacious against established human tumor xenografts in nude mouse models. 15 Two key structural elements of RG7388 were preserved in RO8994. First, it was established that the stereochemical configuration of the pyrrolidine core structure in which the two aryl rings ("A" and "B") adopt a "Trans" orientation was very important for optimal binding to MDM2. 14 The architecture of spiroindolinone-3,3′-pyrrolidine series (as exemplified by MI-219) was first reported by Ding et al. 16−18 Consistent with our findings, this group recently published their latest findings in which the original stereochemistry was found to be unstable and

The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K(i) > 10 microM). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K(i) = 0.001, 0.003, and 0.001 microM for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC(50) = 0.08 microM). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.

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MXCuBE2 is the second-generation evolution of the MXCuBE beamline control software, initially developed and used at ESRF – the European Synchrotron. MXCuBE2 extends, in an intuitive graphical user interface (GUI), the functionalities and data collection methods available to users while keeping all previously available features and allowing for the straightforward incorporation of ongoing and future developments. MXCuBE2 introduces an extended abstraction layer that allows easy interfacing of any kind of macromolecular crystallography (MX) hardware component, whether this is a diffractometer, sample changer, detector or optical element. MXCuBE2 also works in strong synergy with the ISPyB Laboratory Information Management System, accessing the list of samples available for a particular experimental session and associating, either from instructions contained in ISPyB or from user input via the MXCuBE2 GUI, different data collection types to them. The development of MXCuBE2 forms the core of a fruitful collaboration which brings together several European synchrotrons and a software development factory and, as such, defines a new paradigm for the development of beamline control platforms for the European MX user community.