After L858R and ex19del epidermal growth factor receptor (EGFR) mutations, ex20ins mutations are the third most common class of driver-mutations in non-small cell lung cancer (NSCLC). Unfortunately, first-, second-, and third-generation EGFR tyrosine kinase inhibitors (TKIs) are generally ineffective for ex20ins patients due to insufficient mutant activity and selectivity over wild-type EGFR, leading to dose-limiting toxicities. While significant advances in recent years have been made toward identifying potent EGFR ex20ins mutant inhibitors, mutant vs wild-type EGFR selectivity remains a significant challenge. STX-721 (53) is a potent, irreversible inhibitor of the majority of EGFR/HER2 ex20ins mutants and demonstrates excellent mutant vs wild-type selectivity both in vitro and in vivo. STX-721 is currently in phase 1/2 clinical trials for EGFR/HER2 ex20ins-driven NSCLC.

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.

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).

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.

The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.

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.

Achieving selectivity across the human kinome is a major hurdle in kinase inhibitor drug discovery. Assays using active, phosphorylated protein kinases bias hits toward poorly selective inhibitors that bind within the highly conserved adenosine triphosphate (ATP) pocket. Targeting inactive (vs active) kinase conformations offers advantages in achieving selectivity because of their more diversified structures. Kinase cascade assays are typically initiated with target kinases in their unphosphorylated inactive forms, which are activated during the assays. Therefore, these assays are capable of identifying inhibitors that preferentially bind to the unphosphorylated form of the enzyme in addition to those that bind to the active form. We applied this cascade assay to the emerging cancer immunotherapy target hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase that negatively regulates T cell receptor signaling. Using this approach, we discovered an allosteric, inactive conformation-selective triazolopyrimidinone HPK1 inhibitor, compound 1. Compound 1 binds to unphosphorylated HPK1 >24-fold more potently than active HPK1, is not competitive with ATP, and is highly selective against kinases critical for T cell signaling. Furthermore, compound 1 does not bind to the isolated HPK1 kinase domain alone but requires other domains. Together, these data indicate that 1 is an allosteric HPK1 inhibitor that attenuates kinase autophosphorylation by binding to a pocket consisting of residues within and outside of the kinase domain. Our study demonstrates that cascade assays can lead to the discovery of highly selective kinase inhibitors. The triazolopyrimidinone described in this study may represent a privileged chemical scaffold for further development of potent and selective HPK1 inhibitors.

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that plays a critical role in the activation of B cells, macrophages, and osteoclasts. Given the key role of these cell types in the pathology of autoimmune disorders, BTK inhibitors have the potential to improve treatment outcomes in multiple diseases. Herein, we report the discovery and characterization of a novel potent and selective covalent 4-oxo-4,5-dihydro-3H-1-thia-3,5,8-triazaacenaphthylene-2-carboxamide BTK inhibitor chemotype. Compound 27 irreversibly inhibits BTK by targeting a noncatalytic cysteine residue (Cys481) for covalent bond formation. Compound 27 is characterized by selectivity for BTK, potent in vivo BTK occupancy that is sustained after it is cleared from systemic circulation, and dose-dependent efficacy at reducing joint inflammation in a rat collagen-induced arthritis model.

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease characterized by uncontrolled clonal expansion of hematopoietic progenitor cells (myeloid blasts). Disease remission can be achieved with standard induction chemotherapy, but refractory and relapsed disease remains a challenge with a 5-year overall survival rate of approximately 25%. Induction of differentiation is an alternative approach to AML therapy. Dihydroorotate dehydrogenase (DHODH) catalyzes the ubiquinone-mediated oxidation of dihydroorotate (DHO) to orotate in the de novo pyrimidine synthesis pathway. Pre-clinical findings demonstrated that DHODH is a metabolic vulnerability in AML and a new target for differentiation therapy. Given the genetic complexity of myeloid malignancies, future use of DHODH inhibitors in combination with selected antineoplastic agents (e.g., azacitidine) with nonoverlapping mechanisms of action may address specific aspects of the complex pathogenesis of AML and MDS (myelodysplastic syndrome), ultimately improving patients' outcomes.JNJ74856665 is an orally available, potent and selective DHODH inhibitor. JNJ-74856665 inhibited the biochemical activity of human DHODH with a 50% inhibitory concentration (IC50) in the subnanomolar range and in vitro inhibited proliferation of MOLM-13, OCI-AML3, HL60 and THP-1 AML cell lines with low nanomolar IC50 values. Mechanism of action studies using these four cell lines demonstrated induction of CD11b and CD14 differentiation marker mRNA levels. Differentiation was accompanied by cell cycle arrest and apoptosis induction. Importantly, addition of excess uridine to the tissue culture conditions abrogated the anti-proliferative, differentiation and apoptosis inducing activity of JNJ-74856665, indicating on target activity. In vivo, JNJ-74856665 mediated significant activity in decreasing leukemic burden and increasing life span across subcutaneous (MOLM-13 and OCI-AML3) and disseminated (MOLM-13) xenografts, respectively. Pharmacodynamic investigations demonstrated target engagement as evidenced by upregulation of the DHODH substrate dihydroorotate in plasma and induction of differentiation markers in tumor. Combination of JNJ-74856665 with azacitidine in vivo demonstrated lack of antagonism in the anti-leukemic activity.The results described herein demonstrate that JNJ-74856665 exerts potent anti-leukemic activity in vitro and in vivo and warrant further investigation. To that end, a phase 1 clinical trial assessing the safety and efficacy of JNJ-74856665 in patients with AML and MDS is planned to initiate shortly. Citation Format: Christine Pietsch, Scott Kuduk, Xiaochun Zhang, Tammy Bush, Faraz Kazmi, Edgar Jacoby, Zhuming Zhang, Lindsey DeRatt, Weixue Wang, Aaron Patrick, Andrew Steele, Friedericke Pastore, Tony Greway, Nikki Daskalakis, Kathryn Bradford, Kathryn Packman, Gilles Bignan, James Edwards, Ricardo Attar, Yusri Elsayed, Jacqueline Bussolari, Ulrike Philippar. JNJ-74856665, a novel DHODH inhibitor, mediates potent anti-leukemic activity and differentiation in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1256.

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.

G-protein coupled receptor kinase 2 (GRK2), which is upregulated in the failing heart, appears to play a critical role in heart failure (HF) progression in part because enhanced GRK2 activity promotes dysfunction of β-adrenergic signaling and myocyte death. An orally bioavailable GRK2 inhibitor could offer unique therapeutic outcomes that cannot be attained by current heart failure treatments that directly target GPCRs or angiotensin-converting enzyme. Herein, we describe the discovery of a potent, selective, and orally bioavailable GRK2 inhibitor, 8h, through high-throughput screening, hit-to-lead optimization, structure-based design, molecular modelling, synthesis, and biological evaluation. In the cellular target engagement assays, 8h enhances isoproterenol-mediated cyclic adenosine 3′,5′-monophosphate (cAMP) production in HEK293 cells overexpressing GRK2. Compound 8h was further evaluated in a human stem cell-derived cardiomyocyte (HSC-CM) contractility assay and potentiated isoproterenol-induced beating rate in HSC-CMs.

Achieving selectivity across the human kinome is a major hurdle in kinase inhibitor drug discovery. Targeting inactive (vs active) kinase conformations offers advantages in achieving selectivity because of their more diversified structures. Discovery of inactive conformation-selective inhibitors, however, has been hampered partly by the lack of general assay methods. Herein, we show that such inhibitors can be discovered by utilizing kinase cascade assays. This type of assay is initiated with the target kinase in its unphosphorylated, inactive conformation, which is activated during the assay. Inactive conformation-selective inhibitors stabilize the inactive kinase, block activation, and yield reduced kinase activity. We investigate the properties of the assay by mathematical modeling, as well as by proof-of-concept experiments using the BRAF-MEK1 cascade. This study demonstrates effective identification of inactive conformation-selective inhibitors by cascade assays, reveals key factors that impact results, and provides guidelines for successful cascade assay development.

The inside cover picture shows the structure of the isoprenoid biosynthesis enzyme IspH with a newly discovered drug-like inhibitor, a lipophilic barbiturate. IspH catalyzes the formation of (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate, an essential enzyme in isoprenoid biosynthesis in most bacteria, malaria parasites, and plants. The results of spectroscopic studies (including HYSCORE and NRVS) led to a general model for ligand binding to IspH in which ligands bind directly to, or near to, the unique fourth Fe in the 4 Fe−4 S cluster. In silico screening of compound libraries led to new IspH inhibitors with ∼500 nm activity, potential leads for anti-infectives and herbicides. More information can be found in the communication by E. Oldfield et al. on page 914 in Issue 10, 2017 (DOI: 10.1002/cbic.201700052).

The cellular response evoked by a hexanuclear platinum complex, Pt6L4 (1), is reported. Compound 1, a 3-nm octahedral cage formed by self-assembly of six Pt(II) centers and four 2,4,6-tris(4-pyridyl)-1,3,5-triazine ligands (L), exhibits promising in vitro potency against a panel of human cancer cell lines. Unlike classical platinum-based anticancer agents, 1 interacts with DNA in a non-covalent, intercalative manner and promotes DNA condensation. In cancer cells, 1 induces DNA damage, upregulates p53, its phosphorylated form phospho-p53 and its downstream effector, p21, as well as both apoptosis and senescence.

Enzyme des Methylerythritolphosphat‐Weges sind eine attraktive Angriffsstelle für Antiinfektiva. Die letzten beiden Enzyme dieses Biosyntheseweges, IspG und IspH, sind [Fe 4 S 4 ]‐Proteine, die über neuartige Mechanismen 2 H + /2 e − ‐Reduktionen katalysieren und vom Menschen nicht gebildet werden. In diesem Aufsatz fassen wir aktuelle Fortschritte der strukturellen, mechanistischen und inhibitorischen Studien zu diesen beiden Enzymen zusammen. Insbesondere werden mechanistische Vorschläge mit biometallorganischen Zwischenstufen vorgestellt und mit anderen mechanistischen Möglichkeiten verglichen. Auf Substratanaloga basierende Inhibitoren, die durch rationale Überlegungen und das Screening von Substanzbibliotheken entwickelt wurden, werden diskutiert. Die präsentierten Ergebnisse untermauern die biometallorganischen Katalysemechanismen für IspG und IspH und eröffnen Perspektiven für die Entwicklung von Inhibitoren, die auf [Fe 4 S 4 ]‐Cluster in Proteinen zielen.

Enzymes of the methylerythritol phosphate pathway of isoprenoid biosynthesis are attractive anti-infective drug targets. The last two enzymes of this pathway, IspG and IspH, are [Fe4 S4 ] proteins that are not produced by humans and catalyze 2 H(+) / 2 e(-) reductions with novel mechanisms. In this Review, we summarize recent advances in structural, mechanistic, and inhibitory studies of these two enzymes. In particular, mechanistic proposals involving bioorganometallic intermediates are presented, and compared with other mechanistic possibilities. In addition, inhibitors based on substrate analogues as well as developed by rational design and compound-library screening, are discussed. The results presented support bioorganometallic catalytic mechanisms for IspG and IspH, and open up new routes to anti-infective drug design targeting [Fe4 S4 ] clusters in proteins.

IspG and IspH are proteins that are involved in isoprenoid biosynthesis in most bacteria as well as in malaria parasites and are important drug targets. They contain cubane-type 4Fe-4S clusters that are involved in unusual 2H+/2e- reductions. Here, we report the results of electron paramagnetic resonance spectroscopic investigations of the binding of amino- and thiolo-HMBPP (HMBPP=E-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate) IspH substrate-analog inhibitors to both proteins, as well as the binding of HMBPP and an acetylene diphosphate inhibitor, to IspG. The results show that amino-HMBPP binds to reduced IspH by Fe-C π-bonding with the olefinic carbons interacting with the unique 4th Fe in the 4Fe-4S cluster, quite different to the direct Fe-N ligation seen with the oxidized protein. No such π-complex is observed when amino-HMBPP binds to reduced IspG. No EPR signal is observed with IspH in the presence of dithionite and thiolo-HMBPP, suggesting that the 4Fe-4S cluster is not reduced, consistent with the presence of a 420 nm feature in the absorption spectrum (characteristic of an oxidized cluster). However, with IspG, the EPR spectrum in the presence of dithionite and thiolo-HMBPP is very similar to that seen with HMBPP. The binding of HMBPP to IspG was studied using hyperfine sublevel correlation spectroscopy with 17O and 13C labeled samples: the results rule out direct Fe-O bonding and indicate π-bonding. Finally, the binding to IspG of a potent acetylene diphosphate inhibitor was studied by using electron-nuclear double resonance spectroscopy with 13C labeled ligands: the large hyperfine couplings indicate strong Fe-C π-bonding with the acetylenic group. These results illustrate a remarkable diversity in binding behavior for HMBPP-analog inhibitors, opening up new routes to inhibitor design of interest in the context of anti-bacterial and anti-malarial drug discovery, as well as "cubane-type" metallo-biochemistry, in general.

The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases (BMMs) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determine the reductase binding site on the hydroxylase of a BMM enzyme, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism.

The regulatory component (MMOB) of soluble methane monooxygenase (sMMO) has a unique N-terminal tail not found in regulatory proteins of other bacterial multicomponent monooxygenases. This N-terminal tail is indispensable for proper function, yet its solution structure and role in catalysis remain elusive. Here, by using double electron-electron resonance (DEER) spectroscopy, we show that the oxidation state of the hydroxylase component, MMOH, modulates the conformation of the N-terminal tail in the MMOH-2MMOB complex, which in turn facilitates catalysis. The results reveal that the N-terminal tail switches from a relaxed, flexible conformational state to an ordered state upon MMOH reduction from the diiron(III) to the diiron(II) state. This observation suggests that some of the crystallographically observed allosteric effects that result in the connection of substrate ingress cavities in the MMOH-2MMOB complex may not occur in solution in the diiron(III) state. Thus, O2 may not have easy access to the active site until after reduction of the diiron center. The observed conformational change is also consistent with a higher binding affinity of MMOB to MMOH in the diiron(II) state, which may allow MMOB to displace more readily the reductase component (MMOR) from MMOH following reduction.