Discovery and Optimization of DNA Gyrase and Topoisomerase IV Inhibitors with Potent Activity against Fluoroquinolone-Resistant Gram-Positive Bacteria.

Herein, we describe the discovery and optimization of a novel series that inhibits bacterial DNA gyrase and topoisomerase IV via binding to, and stabilization of, DNA cleavage complexes. Optimization of this series led to the identification of compound 25, which has potent activity against Gram-positive bacteria, a favorable in vitro safety profile, and excellent in vivo pharmacokinetic properties. Compound 25 was found to be efficacious against fluoroquinolone-sensitive Staphylococcus aureus infection in a mouse thigh model at lower doses than moxifloxacin. An X-ray crystal structure of the ternary complex formed by topoisomerase IV from Klebsiella pneumoniae, compound 25, and cleaved DNA indicates that this compound does not engage in a water-metal ion bridge interaction and forms no direct contacts with residues in the quinolone resistance determining region (QRDR). This suggests a structural basis for the reduced impact of QRDR mutations on antibacterial activity of 25 compared to fluoroquinolones.

Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria.

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region.

A polyomavirus peptide binds to the capsid VP1 pore and has potent antiviral activity against BK and JC polyomaviruses.

In pursuit of therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen-residue peptide binds to major structural protein VP1 with single-digit nanomolar affinity. Alanine-scanning of the peptide identified three key residues, substitution of each of which results in ~1000 fold loss of binding affinity with a concomitant reduction in antiviral activity. Structural studies demonstrate specific binding of the peptide to the pore of pentameric VP1. Cell-based assays demonstrate nanomolar inhibition (EC50) of BKV infection and suggest that the peptide acts early in the viral entry pathway. Homologous peptide exhibits similar binding to JC polyomavirus VP1 and inhibits infection with similar potency to BKV in a model cell line. Lastly, these studies validate targeting the VP1 pore as a novel strategy for the development of anti-polyomavirus agents.

Author Correction: Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Discovery and optimization of novel pyridines as highly potent and selective glycogen synthase kinase 3 inhibitors.

Glycogen synthase kinase-3 plays an essential role in multiple biochemical pathways in the cell, particularly in regards to energy regulation. As such, Glycogen synthase kinase-3 is an attractive target for pharmacological intervention in a variety of disease states, particularly non-insulin dependent diabetes mellitus. However, due to homology with other crucial kinases, such as the cyclin-dependent protein kinase CDC2, developing compounds that are both potent and selective is challenging. A novel series of derivatives of 5-nitro-N2-(2-(pyridine-2ylamino)ethyl)pyridine-2,6-diamine were synthesized and have been shown to potently inhibit glycogen synthase kinase-3 (GSK3). Potency in the low nanomolar range was obtained along with remarkable selectivity. The compounds activate glycogen synthase in insulin receptor-expressing CHO-IR cells and in primary rat hepatocytes, and have acceptable pharmacokinetics and pharmacodynamics to allow for oral dosing. The X-ray co-crystal structure of human GSK3-ß in complex with compound 2 is reported and provides insights into the structural determinants of the series responsible for its potency and selectivity.

Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex.

The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.

Harnessing the anti-cancer natural product nimbolide for targeted protein degradation.

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.

First Crystal Structure of a Nonstructural Hepatitis E Viral Protein Identifies a Putative Novel Zinc-Binding Protein.

Hepatitis E virus (HEV) is a 7.2-kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF1 to ORF3. All nonstructural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression, and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a nonclassical, potentially catalytic zinc-binding motif. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome. We present analysis for the first time of this identified nonstructural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology.IMPORTANCE Hepatitis E virus (HEV) is an emerging virus found predominately in developing countries; it causes an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the nonstructural proteins of HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. Here we present the first structural and biophysical characterization of an HEV nonstructural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease.

High-Confidence Protein-Ligand Complex Modeling by NMR-Guided Docking Enables Early Hit Optimization.

Structure-based drug design is an integral part of modern day drug discovery and requires detailed structural characterization of protein-ligand interactions, which is most commonly performed by X-ray crystallography. However, the success rate of generating these costructures is often variable, in particular when working with dynamic proteins or weakly binding ligands. As a result, structural information is not routinely obtained in these scenarios, and ligand optimization is challenging or not pursued at all, representing a substantial limitation in chemical scaffolds and diversity. To overcome this impediment, we have developed a robust NMR restraint guided docking protocol to generate high-quality models of protein-ligand complexes. By combining the use of highly methyl-labeled protein with experimentally determined intermolecular distances, a comprehensive set of protein-ligand distances is generated which then drives the docking process and enables the determination of the correct ligand conformation in the bound state. For the first time, the utility and performance of such a method is fully demonstrated by employing the generated models for the successful, prospective optimization of crystallographically intractable fragment hits into more potent binders.

Synthesis, Binding Mode, and Antihyperglycemic Activity of Potent and Selective (5-Imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridylamino)ethyl]amine Inhibitors of Glycogen Synthase Kinase 3.

In an effort to identify new antidiabetic agents, we have discovered a novel family of (5-imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridylamino)ethyl]amine analogues which are inhibitors of human glycogen synthase kinase 3 (GSK3). We developed efficient synthetic routes to explore a wide variety of substitution patterns and convergently access a diverse array of analogues. Compound 1 (CHIR-911, CT-99021, or CHIR-73911) emerged from an exploration of heterocycles at the C-5 position, phenyl groups at C-4, and a variety of differently substituted linker and aminopyridine moieties attached at the C-2 position. These compounds exhibited GSK3 IC50s in the low nanomolar range and excellent selectivity. They activate glycogen synthase in insulin receptor-expressing CHO-IR cells and primary rat hepatocytes. Evaluation of lead compounds 1 and 2 (CHIR-611 or CT-98014) in rodent models of type 2 diabetes revealed that single oral doses lowered hyperglycemia within 60 min, enhanced insulin-stimulated glucose transport, and improved glucose disposal without increasing insulin levels.

Structural basis for therapeutic inhibition of influenza A polymerase PB2 subunit.

Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11-13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins. VX-787 is an inhibitor of the influenza A virus pre-mRNA cap-binding protein PB2. This clinical stage compound was shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, the binding of this molecule in the context of an extended form of the PB2 subunit has remained elusive. Here we generated a collection of PB2 truncations to identify a PB2 protein representative of its structure in the viral heterotrimeric protein. We present the crystal structure of VX-787 bound to a PB2 construct that recapitulates VX-787's biological antiviral activity in vitro. This co-structure reveals more extensive interactions than previously identified and provides insight into the observed resistance profile, affinity, binding kinetics, and conformational rearrangements induced by VX-787.

Inhibition of prenylated KRAS in a lipid environment.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.

Rationally Designed PI3Kα Mutants to Mimic ATR and Their Use to Understand Binding Specificity of ATR Inhibitors.

ATR, a protein kinase in the PIKK family, plays a critical role in the cell DNA-damage response and is an attractive anticancer drug target. Several potent and selective inhibitors of ATR have been reported showing significant antitumor efficacy, with most advanced ones entering clinical trials. However, due to the absence of an experimental ATR structure, the determinants contributing to ATR inhibitors' potency and specificity are not well understood. Here we present the mutations in the ATP-binding site of PI3Kα to progressively transform the pocket to mimic that of ATR. The generated PI3Kα mutants exhibit significantly improved affinity for selective ATR inhibitors in multiple chemical classes. Furthermore, we obtained the X-ray structures of the PI3Kα mutants in complex with the ATR inhibitors. The crystal structures together with the analysis on the inhibitor affinity profile elucidate the roles of individual amino acid residues in the binding of ATR inhibitors, offering key insights for the binding mechanism and revealing the structure features important for the specificity of ATR inhibitors. The ability to obtain structural and binding data for these PI3Kα mutants, together with their ATR-like inhibitor binding profiles, makes these chimeric PI3Kα proteins valuable model systems for structure-based inhibitor design.

Discovery and Molecular Basis of a Diverse Set of Polycomb Repressive Complex 2 Inhibitors Recognition by EED.

Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.

Structure-Guided Design of EED Binders Allosterically Inhibiting the Epigenetic Polycomb Repressive Complex 2 (PRC2) Methyltransferase.

PRC2 is a multisubunit methyltransferase involved in epigenetic regulation of early embryonic development and cell growth. The catalytic subunit EZH2 methylates primarily lysine 27 of histone H3, leading to chromatin compaction and repression of tumor suppressor genes. Inhibiting this activity by small molecules targeting EZH2 was shown to result in antitumor efficacy. Here, we describe the optimization of a chemical series representing a new class of PRC2 inhibitors which acts allosterically via the trimethyllysine pocket of the noncatalytic EED subunit. Deconstruction of a larger and complex screening hit to a simple fragment-sized molecule followed by structure-guided regrowth and careful property modulation were employed to yield compounds which achieve submicromolar inhibition in functional assays and cellular activity. The resulting molecules can serve as a simplified entry point for lead optimization and can be utilized to study this new mechanism of PRC2 inhibition and the associated biology in detail.

Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit.

Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2.

Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo.

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.

Discovery of 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398), a potent and selective inhibitor of the fibroblast growth factor receptor family of receptor tyrosine kinase.

A novel series of N-aryl-N'-pyrimidin-4-yl ureas has been optimized to afford potent and selective inhibitors of the fibroblast growth factor receptor tyrosine kinases 1, 2, and 3 by rationally designing the substitution pattern of the aryl ring. On the basis of its in vitro profile, compound 1h (NVP-BGJ398) was selected for in vivo evaluation and showed significant antitumor activity in RT112 bladder cancer xenografts models overexpressing wild-type FGFR3. These results support the potential therapeutic use of 1h as a new anticancer agent.

Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer.

Phosphoinositide-3-kinases (PI3Ks) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clinical trials for the treatment of cancer.

Design and synthesis of benzoazepin-2-one analogs as allosteric binders targeting the PIF pocket of PDK1.

A novel series of benzoazepin-2-ones were designed and synthesized targeting the PIF pocket of AGC protein kinases, among which a series of thioether-linked benzoazepin-2-ones were discovered to bind to the PIF pocket of 3-phosphoinositide-dependent kinase-1 (PDK1), and to displace the PIF peptide with an EC(50) values in the lower micromolar range. The structure-activity relationships (SARs) of the linker region, tail region, and distal region were explored to further optimize these novel binders which target the PIF pocket of PDK1. When tested in an in vitro PDK1 enzymatic assay using a peptide substrate, the benzodiazepin-2-ones increased the activity of the enzyme in a concentration-dependent fashion, indicating these compounds act as PDK1 allosteric activators. These new compounds may be further developed as therapeutic agents for the treatment of diseases where the PDK1-mediated AGC protein kinases are dysregulated.