Discovery of JNJ-74856665: A Novel Isoquinolinone DHODH Inhibitor for the Treatment of AML.

Acute myelogenous leukemia (AML), a heterogeneous disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS).

Discovery of Alternative Binding Poses through Fragment-Based Identification of DHODH Inhibitors.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival. Recently, DHODH has been identified as a potential target for acute myeloid leukemia therapy. Herein, we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode. Additionally, these compounds had low P-gp efflux ratios, allowing for applications where exposure to the brain would be required.

N-Heterocyclic 3-Pyridyl Carboxamide Inhibitors of DHODH for the Treatment of Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML), a disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway; however, small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes. Using virtual screening and structure-based drug design approaches, a new series of N-heterocyclic 3-pyridyl carboxamide DHODH inhibitors were discovered. Two lead compounds, 19 and 29, have potent biochemical and cellular DHODH activity, favorable physicochemical properties, and efficacy in a preclinical model of AML.

A carboxylic acid isostere screen of the DHODH inhibitor Brequinar.

Dihydroorotate dehydrogenase (DHODH) enzymatic activity impacts many aspects critical to cell proliferation and survival. Recently, DHODH has been identified as a target for acute myeloid differentiation therapy. In preclinical models of AML, the DHODH inhibitor Brequinar (BRQ) demonstrated potent anti-leukemic activity. Herein we describe a carboxylic acid isostere study of Brequinar which revealed a more potent non-carboxylic acid derivative with improved cellular potency and good pharmacokinetic properties.

Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling.

The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.

Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo)kinases upon Binding Small Molecules.

Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.

Discovery of dihydroisoquinolinone derivatives as novel inhibitors of the p53-MDM2 interaction with a distinct binding mode.

Blocking the interaction between the p53 tumor suppressor and its regulatory protein MDM2 is a promising therapeutic concept under current investigation in oncology drug research. We report here the discovery of the first representatives of a new class of small molecule inhibitors of this protein-protein interaction: the dihydroisoquinolinones. Starting from an initial hit identified by virtual screening, a derivatization program has resulted in compound 11, a low nanomolar inhibitor of the p53-MDM2 interaction showing significant cellular activity. Initially based on a binding mode hypothesis, this effort was then guided by a X-ray co-crystal structure of MDM2 in complex with one of the synthesized analogs. The X-ray structure revealed an unprecedented binding mode for p53-MDM2 inhibitors.

Structural investigation of B-Raf paradox breaker and inducer inhibitors.

The V600E missense mutation in B-Raf kinase leads to an anomalous regulation of the MAPK pathway, uncontrolled cell proliferation, and initiation of tumorigenesis. While the ATP-competitive B-Raf inhibitors block the MAPK pathway in B-Raf mutant cells, they induce conformational changes to wild-type B-Raf kinase domain leading to heterodimerization with C-Raf causing a paradoxical hyperactivation of MAPK pathway. A new class of inhibitors (paradox breakers) has been developed that inhibit B-Raf(V600E) activity without agonistically affecting the MAPK pathway in wild-type B-Raf cells. In this study, we explore the structural, conformational, and cellular effects on the B-Raf kinase domain upon binding of paradox breakers and inducers. Our results indicate that a subtle structural difference between paradox inducers and breakers leads to significant conformational differences when complexed with B-Raf. This study provides a novel insight into the activation of B-Raf by ATP-competitive inhibitors and can aid in the design of more potent and selective inhibitors without agonistic function.

Knowledge-based virtual screening: application to the MDM4/p53 protein-protein interaction.

Chemogenomics knowledge-based drug discovery approaches aim to extract the knowledge gained from one target and to apply it for the discovery of ligands and hopefully drugs of a new target which is related to the parent target by homology or conserved molecular recognition. Herein, we demonstrate the potential of knowledge-based virtual screening by applying it to the MDM4-p53 protein-protein interaction where the MDM2-p53 protein-protein interaction constitutes the parent reference system; both systems are potentially relevant to cancer therapy. We show that a combination of virtual screening methods, including homology based similarity searching, QSAR (Quantitative Structure-Activity Relationship) methods, HTD (High Throughput Docking), and UNITY pharmacophore searching provide a successful approach to the discovery of inhibitors. The virtual screening hit list is of the magnitude of 50,000 compounds picked from the corporate compound library of approximately 1.2 million compounds. Emphasis is placed on the facts that such campaigns are only feasible because of the now existing HTCP (High throughput Cherry-Picking) automation systems in combination with robust MTS (Medium Throughput Screening) fluorescence-based assays. Given that the MDM2-p53 system constitutes the reference system, it is not surprising that significantly more and stronger hits are found for this interaction compared to the MDM4-p53 system. Novel, selective and dual hits are discovered for both systems. A hit rate analysis will be provided compared to the full HTS (High-throughput Screening).

A small-molecule dengue virus entry inhibitor.

The incidence of dengue fever epidemics has increased dramatically over the last few decades. However, no vaccine or antiviral therapies are available. Therefore, the need for safe and effective antiviral drugs has become imperative. The entry of dengue virus into a host cell is mediated by its major envelope (E) protein. The crystal structure of the E protein reveals a hydrophobic pocket that is presumably important for low-pH-mediated membrane fusion. High-throughput docking with this hydrophobic pocket was performed, and hits were evaluated in cell-based assays. Compound 6 was identified as one of the inhibitors and had an average 50% effective concentration of 119 nM against dengue virus serotype 2 in a human cell line. Mechanism-of-action studies demonstrated that compound 6 acts at an early stage during dengue virus infection. It arrests dengue virus in vesicles that colocalize with endocytosed dextran and inhibits NS3 expression. The inhibitors described in this report can serve as molecular probes for the study of the entry of flavivirus into host cells.

Docking of ATP to Ca-ATPase: considering protein domain motions.

Most standard molecular docking algorithms take into account only ligand flexibility, while numerous studies demonstrate that receptor flexibility may be also important. While some efficient methods have been proposed to take into account local flexibility of protein side chains, the influence of large-scale domain motions on the docking results still represents a challenge for computational methods. In this work we compared the results of ATP docking to different models of Ca-ATPase: crystallographic apo- and holo-forms of the enzyme as well as "flexible" target models generated via molecular dynamics (MD) simulations in water. MD simulations were performed for two different apo-forms and one holo-form of Ca2+-ATPase and reveal large-scale domain motions of type "closure", which is consistent with experimental structures. Docking to a set of MD-conformers yielded correct solutions with ATP bound in both domains regardless of the starting Ca2+-ATPase structure. Also, special attention was paid to proper ranking of docking solutions and some particular features of different scoring functions and their applicability for the model of "flexible" receptor. Particularly, the results of docking ATP were ranked by a scoring criterion specially designed to estimate ATP-protein interactions. This criterion includes stacking and hydrophobic interactions characteristic of ATP-protein complexes. The performance of this ligand-specific scoring function was considerably better than that of a standard scoring function used in the docking algorithm.

Complementarity of hydrophobic properties in ATP-protein binding: a new criterion to rank docking solutions.

ATP is an important substrate of numerous biochemical reactions in living cells. Molecular recognition of this ligand by proteins is very important for understanding enzymatic mechanisms. Considerable insight into the problem may be gained via molecular docking simulations. At the same time, standard docking protocols are often insufficient to predict correct conformations for protein-ATP complexes. Thus, in most cases the native-like solutions can be found among the docking poses, but current scoring functions have only limited ability to discriminate them from false positives. To improve the selection of correct docking solutions obtained with the GOLD software, we developed a new ranking criterion specific for ATP-protein binding. The method is based on detailed analysis of the intermolecular interactions in 40 high-resolution 3D structures of ATP-protein complexes (the training set). We found that the most important factors governing this recognition are hydrogen-bonding, stacking between adenine and aromatic protein residues, and hydrophobic contacts between adenine and protein residues. To address the latter, we applied the formalism of 3D molecular hydrophobicity potential. The results obtained were used to construct an ATP-oriented scoring criterion as a linear combination of the terms describing these intermolecular interactions. The criterion was then validated using the test set of 10 additional ATP-protein complexes. As compared with the standard scoring functions, the new ranking criterion significantly improved the selection of correct docking solutions in both sets and allowed considerable enrichment at the top of the list containing docking poses with correct solutions.