Efficiently driving protein-based fragment screening and lead discovery using two-dimensional NMR.

Fragment-based drug discovery (FBDD) and validation of small molecule binders using NMR spectroscopy is an established and widely used method in the early stages of drug discovery. Starting from a library of small compounds, ligand- or protein-observed NMR methods are employed to detect binders, typically weak, that become the starting points for structure-activity relationships (SAR) by NMR. Unlike the more frequently used ligand-observed 1D NMR techniques, protein-observed 2D 1H-15N or 1H-13C heteronuclear correlation (HSQC or HMQC) methods offer insights that include the mechanism of ligand engagement on the target and direct binding affinity measurements in addition to routine screening. We hereby present the development of a set of software tools within the MestReNova (Mnova) package for analyzing 2D NMR for FBDD and hit validation purposes. The package covers three main tasks: (1) unsupervised profiling of raw data to identify outlier data points to exclude in subsequent analyses; (2) batch processing of single-point spectra to identify and rank binders based on chemical shift perturbations or spectral peak intensity changes; and (3) batch processing of multiple titration series to derive binding affinities (KD) by tracing the changes in peak locations or measuring global spectral changes. Toward this end, we implemented and evaluated a set of algorithms for automated peak tracing, spectral binning, and variance analysis by PCA, and a new tool for spectral data intensity comparison using ECHOS. The accuracy and speed of the tools are demonstrated on 2D NMR binding data collected on ligands used in the development of potential inhibitors of the anti-apoptotic MCL-1 protein.

Identification and structure-based drug design of cell-active inhibitors of interleukin 17A at a novel C-terminal site.

Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein-protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (< 50 nM) and showed functional inhibition of IL17A-induced cellular signaling (IC50~1 µM). We also describe a fluorescence-based probe molecule suitable to discern/screen for additional molecules binding in this C-terminal site.

Insights into the Conformation and Self-Association of a Concentrated Monoclonal Antibody using Isothermal Chemical Denaturation and Nuclear Magnetic Resonance.

The purpose of this investigation was to highlight the utility of nuclear magnetic resonance (NMR) as a multi-attribute method for the characterization of therapeutic antibodies. In this case study, we compared results from isothermal chemical denaturation (ICD) and NMR with standard methods to relate conformational states of a model monoclonal antibody (mAb1) with protein-protein interactions (PPI) that lead to self - association in concentrated solutions. The increase in aggregation rate and relative viscosity for mAb1 was found to be both concentration and pH dependent. The free energy of unfolding (∆G°) from ICD and thermal analysis in dilute solutions indicated that although the native state predominated between pH 4 - pH 7, it was disrupted at the CH2 and unfolded noncooperatively under acidic conditions. One-dimensional (1D) 1H NMR and two-dimensional (2D) 13C-1H NMR performed, in concentrated solutions, confirmed that PPI between pH 4-7 occurred while mAb1 was in the native state. NMR corroborated that mAb1 maintained a dominant native state at formulation-relevant conditions at the tested pH range, had increased global molecular tumbling dynamics at lower pH and confirmed increased PPI at higher pH conditions. This report aligns and compares typical characterization of an IgG1 with assessment of structure by NMR and provided a more precise assessment and deeper insight into the conformation of an IgG1 in concentrated solutions.

Development of Orally Efficacious Allosteric Inhibitors of TNFα via Fragment-Based Drug Design.

Tumor necrosis factor α (TNFα) is a soluble cytokine that is directly involved in systemic inflammation through the regulation of the intracellular NF-κB and MAPK signaling pathways. The development of biologic drugs that inhibit TNFα has led to improved clinical outcomes for patients with rheumatoid arthritis and other chronic autoimmune diseases; however, TNFα has proven to be difficult to drug with small molecules. Herein, we present a two-phase, fragment-based drug discovery (FBDD) effort in which we first identified isoquinoline fragments that disrupt TNFα ligand-receptor binding through an allosteric desymmetrization mechanism as observed in high-resolution crystal structures. The second phase of discovery focused on the de novo design and optimization of fragments with improved binding efficiency and drug-like properties. The 3-indolinone-based lead presented here displays oral, in vivo efficacy in a mouse glucose-6-phosphate isomerase (GPI)-induced paw swelling model comparable to that seen with a TNFα antibody.

NMR-based fragment screening and lead discovery accelerated by principal component analysis.

Protein-based NMR spectroscopy has proven to be a very robust method for finding fragment leads to protein targets. However, one limitation of protein-based NMR is that the data acquisition and analysis can be time consuming. In order to streamline the scoring of protein-based NMR fragment screening data and the determination of ligand affinities using 2D NMR experiments we have developed a data analysis workflow based on principal component analysis (PCA) within the TREND NMR Pro software package. We illustrate this using four different proteins and sets of ligands which interact with these proteins over a range of affinities. Also, these PCA-based methods can be successfully applied even to systems where ligand binding to target proteins is in intermediate or even slow exchange on the NMR time scale. Finally, these methods will work for scoring of fragment binding data using protein spectra that are either highly overlapped or lower in resolution.

Fragment-Based Discovery of an Apolipoprotein E4 (apoE4) Stabilizer.

Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer's disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.

Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain - A fragment based approach.

The tandem TUDOR domains present in the non-catalytic C-terminal half of the KDM4A, 4B and 4C enzymes play important roles in regulating their chromatin localizations and substrate specificities. They achieve this regulatory role by binding to different tri-methylated lysine residues on histone H3 (H3-K4me3, H3-K23me3) and histone H4 (H4-K20me3) depending upon the specific chromatin environment. In this work, we have used a 2D-NMR based fragment screening approach to identify a novel fragment (1a), which binds to the KDM4A-TUDOR domain and shows modest competition with H3-K4me3 binding in biochemical as well as in vitro cell based assays. A co-crystal structure of KDM4A TUDOR domain in complex with 1a shows that the fragment binds stereo-specifically to the methyl lysine binding pocket forming a network of strong hydrogen bonds and hydrophobic interactions. We anticipate that the fragment 1a can be further developed into a novel allosteric inhibitor of the KDM4 family of enzymes through targeting their C-terminal tandem TUDOR domain.

Fragment-based discovery of a potent NAMPT inhibitor.

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor.

Fragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain Inhibitors.

Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 µM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.

Methylpyrrole inhibitors of BET bromodomains.

An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma.

The EED protein-protein interaction inhibitor A-395 inactivates the PRC2 complex.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8.

SETD8 is a histone H4-K20 methyltransferase that plays an essential role in the maintenance of genomic integrity during mitosis and in DNA damage repair, making it an intriguing target for cancer research. While some small molecule inhibitors for SETD8 have been reported, the structural binding modes for these inhibitors have not been revealed. Using the complex structure of the substrate peptide bound to SETD8 as a starting point, different natural and unnatural amino acid substitutions were tested, and a potent (Ki 50 nM, IC50 0.33 µM) and selective norleucine containing peptide inhibitor has been obtained.

Structure-guided design of a series of MCL-1 inhibitors with high affinity and selectivity.

Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein we report a series of MCL-1 inhibitors that emanated from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compounds from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compound 30b rapidly induced caspase activation with associated loss in cell viability. The small molecules described herein thus comprise effective tools for studying MCL-1 biology.

Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity.

A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.

Discovery and development of potent and selective inhibitors of histone methyltransferase g9a.

G9a is a histone lysine methyltransferase responsible for the methylation of histone H3 lysine 9. The discovery of A-366 arose from a unique diversity screening hit, which was optimized by incorporation of a propyl-pyrrolidine subunit to occupy the enzyme lysine channel. A-366 is a potent inhibitor of G9a (IC50: 3.3 nM) with greater than 1000-fold selectivity over 21 other methyltransferases.

Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts.

Cracking the molecular weight barrier: fragment screening of an aminotransferase using an NMR-based functional assay.

NMR-based screening of protein targets has become a well established part of the drug discovery process especially with respect to fragments. However, as target size increases the two-dimensional spectra typically used for such screening become more crowded due to the increased number of signals, and the individual signals broaden due to the decreased rotational correlation time of the protein. Here we present an NMR-based functional assay for the branched-chain aminotransferase BCATc, a dimer with a total molecular weight of 88 kDa, which overcomes the limitations of the typical protein-based NMR screening method. BCATc is involved in glutamate production in the brain and is a therapeutic target for neuronal disorders involving a glutamatergic mechanism. Several fragments which inhibit BCATc were discovered using this assay and these may serve as novel cores for the development of potent BCATc inhibitors.

Fragment-based lead discovery: challenges and opportunities.

Fragment-based lead discovery has undergone remarkable changes over the last 15 years. During this time, the pharmaceutical industry has changed dramatically as well, and continued evolution of the industry is assured. These changes present many challenges but also several opportunities for executing fragment-based drug design. This article will explore some of the more significant changes in the industry and how they may affect future discovery efforts related to fragment-based initiatives.