Characterization of RNA driven structural changes in full length RIG-I leading to its agonism or antagonism.

RIG-I (retinoic acid inducible gene-I) can sense subtle differences between endogenous and viral RNA in the cytoplasm, triggering an anti-viral immune response through induction of type I interferons (IFN) and other inflammatory mediators. Multiple crystal and cryo-EM structures of RIG-I suggested a mechanism in which the C-terminal domain (CTD) is responsible for the recognition of viral RNA with a 5'-triphoshate modification, while the CARD domains serve as a trigger for downstream signaling, leading to the induction of type I IFN. However, to date contradicting conclusions have been reached around the role of ATP in the mechanism of the CARD domains ejection from RIG-I's autoinhibited state. Here we present an application of NMR spectroscopy to investigate changes induced by the binding of 5'-triphosphate and 5'-OH dsRNA, both in the presence and absence of nucleotides, to full length RIG-I with all its methionine residues selectively labeled (Met-[ϵ-13CH3]). With this approach we were able to identify residues on the CTD, helicase domain, and CARDs that served as probes to sense RNA-induced conformational changes in those respective regions. Our results were analyzed in the context of either agonistic or antagonistic RNAs, by and large supporting a mechanism proposed by the Pyle Lab in which CARD release is primarily dependent on the RNA binding event.

Combining NMR and X-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective BACE-1 inhibitors.

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.

Discovery of MK-1454: A Potent Cyclic Dinucleotide Stimulator of Interferon Genes Agonist for the Treatment of Cancer.

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

Rapid Evolution of a Fragment-like Molecule to Pan-Metallo-Beta-Lactamase Inhibitors: Initial Leads toward Clinical Candidates.

With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by 11, discovered earlier as B. fragilis metallo-beta-lactamase inhibitors, was selected for in silico virtual screening. From these efforts, compound 12 was identified with activity against NDM-1 only. Initial exploration on metal binding design followed by structure-guided optimization led to the discovery of a series of compounds represented by 23 with a pan MBL inhibition profile. In in vivo studies, compound 23 in combination with imipenem (IPM) robustly lowered the bacterial burden in a murine infection model and became the lead for the invention of MBLI clinical candidates.

An orally available non-nucleotide STING agonist with antitumor activity.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

NMR Binding and Functional Assays for Detecting Inhibitors of S. aureus MnaA.

Nonessential enzymes in the staphylococcal wall teichoic acid (WTA) pathway serve as highly validated ß-lactam potentiation targets. MnaA (UDP-GlcNAc 2-epimerase) plays an important role in an early step of WTA biosynthesis by providing an activated form of ManNAc. Identification of a selective MnaA inhibitor would provide a tool to interrogate the contribution of the MnaA enzyme in the WTA pathway as well as serve as an adjuvant to restore ß-lactam activity against methicillin-resistant Staphylococcus aureus (MRSA). However, development of an epimerase functional assay can be challenging since both MnaA substrate and product (UDP-GlcNAc/UDP-ManNAc) share an identical molecular weight. Herein, we developed a nuclear magnetic resonance (NMR) functional assay that can be combined with other NMR approaches to triage putative MnaA inhibitors from phenotypic cell-based screening campaigns. In addition, we determined that tunicamycin, a potent WTA pathway inhibitor, inhibits both S. aureus MnaA and a functionally redundant epimerase, Cap5P.

NMR screening in fragment-based drug design: a practical guide.

Fragment-based drug design (FBDD) comprises both fragment-based screening (FBS) to find hits and elaboration of these hits to lead compounds. Typical fragment hits have lower molecular weight (<300-350 Da) and lower initial potency but higher ligand efficiency when compared to those from high-throughput screening. NMR spectroscopy has been widely used for FBDD since it identifies and localizes the binding site of weakly interacting hits on the target protein. Here we describe ligand-based NMR methods for hit identification from fragment libraries and for functional cross-validation of primary hits.

Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2.

The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d(2Delta)NS3h) by NMR and circular dichroism. Binding of dsDNA to d(2Delta)NS3h induces a local unfolding of helix (alpha(3)), which includes residues of conserved helicase motif VI (Q(460)RxxRxxR(467)), and strands (beta(1) and beta(8)) from the central beta-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d(2Delta)NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg-Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function.

Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications.

The type I interferon alpha family consists of small proteins that have clinically important anti-infective and anti-tumor activity. Interferon alpha-2b (Intron A) combination therapy with ribavirin is the current standard of care for the treatment of chronic hepatitis C virus infection. A drawback to the therapy however, is the short serum half-life and rapid clearance of the interferon alpha protein. Schering-Plough has developed a semi-synthetic form of Intron A by attaching a 12-kDa mono-methoxy polyethylene glycol to the protein (PEG Intron) which fulfills the requirements of a long-acting interferon alpha protein while providing significant clinical benefits. A detailed physicochemical and biological characterization of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Though pegylation appeared to decrease the specific activity of the interferon alpha-2b protein, the potency of PEG Intron, independent of protein concentration, was comparable to the Intron A standard at both the molecular and cellular level. Importantly, PEG Intron has demonstrated an enhanced pharmacokinetic profile in both animal and human studies. Recently, PEG Intron in combination with ribavirin has been shown to be very effective in reducing hepatitis C viral load and maintaining effective sustained viral suppression in patients. Because of the improved clinical benefits, it is anticipated that the PEG Intron plus ribavirin combination therapy will become the new standard of care for the treatment of chronic hepatitis C.

Structure, biology, and therapeutic implications of pegylated interferon alpha-2b.

Derivatization of protein-based therapeutics with polyethylene glycol (pegylation) can often improve pharmacokinetic and pharmacodynamic properties of the proteins and thereby, improve efficacy and minimize dosing frequency. This review will provide an overview of pegylation technology and pegylated protein-based drugs being used or investigated clinically. The novel therapeutic, PEG Intron(R), formed by attaching a 12-kDa mono-methoxy polyethylene glycol (PEG) to the interferon alpha-2b protein, will be discussed in detail in terms of its structure, biological activities, pharmacokinetic properties, and clinical efficacy for the treatment of chronic hepatitis C. Detailed physicochemical and biological characterization studies of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Pegylation of Intron A at pH 6.5 results in a mixture of > or = 95% mono-pegylated isoforms with the predominant species (approximately 50%) derivatized to the His(34) residue with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, as well as serine, tyrosine, and another histidine residue. The anti-viral activity for each pegylated isomer showed that the highest specific activity (37%) was associated with the His(34)-pegylated isomer. Though pegylation decreases the specific activity of the interferon alpha-2b protein in vitro, the potency of PEG Intron was comparable to the Intron A standard at both the molecular and cellular level. The substituted IFN had an enhanced pharmacokinetic profile in both animal and human studies, and, when combined with ribavirin, was very effective in reducing hepatitis C viral load and maintaining sustained viral suppression in patients.

NMR-based approaches for lead discovery.

NMR methods have long been used for studying molecular interactions. In the last few years, various NMR approaches have been developed to aid lead discovery. These involve different NMR screening methods to identify initial compounds, which often bind only weakly (in the micro- to millimolar range) to the drug target. Intelligent and focused follow-up strategies enable the development of these compounds into potent, submicromolar drug-like inhibitors for use as leads in drug discovery projects. NMR can be used as both a remarkably reliable screening tool and a structural tool; thus, this technique has unique opportunities for lead discovery.

Non-peptidic small-molecule inhibitors of the single-chain hepatitis C virus NS3 protease/NS4A cofactor complex discovered by structure-based NMR screening.

NMR-based screening of a customized fragment library identified 16 small-molecule hits that bind weakly (K(D) approximately 100 microM to 10 mM) to substrate binding sites of the NS4A-bound NS3 protease of the hepatitis C virus (HCV). Analogues for five classes of NMR hits were evaluated by a combination of NMR and biochemical data yielding SAR and, in most cases, optimized hits with improved potencies (K(D) approximately K(I) approximately 40 microM to 1 mM). NMR chemical shift perturbation data were used to establish the binding location and orientation of the active site directed scaffolds in these five analogue series. Two of these scaffolds, which bind the enzyme at the proximal S1-S3 and S2' substrate binding sites, were linked together producing competitive inhibitors of the HCV NS3 protease with potencies in the micromolar range. This example illustrates that the low molecular weight scaffolds discovered from structure-based NMR screening can be optimized with focused structure-guided chemistry to produce potent nonpeptidic small-molecule inhibitors of the HCV NS3 protease.

Effective progression of nuclear magnetic resonance-detected fragment hits.

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade as an alternate lead generation tool to HTS approaches. Several compounds have now progressed into the clinic which originated from a fragment-based approach, demonstrating the utility of this emerging field. While fragment hit identification has become much more routine and may involve different screening approaches, the efficient progression of fragment hits into quality lead series may still present a major bottleneck for the broadly successful application of FBDD. In our laboratory, we have extensive experience in fragment-based NMR screening (SbN) and the subsequent iterative progression of fragment hits using structure-assisted chemistry. To maximize impact, we have applied this approach strategically to early- and high-priority targets, and those struggling for leads. Its application has yielded a clinical candidate for BACE1 and lead series in about one third of the SbN/FBDD projects. In this chapter, we will give an overview of our strategy and focus our discussion on NMR-based FBDD approaches.

Discovery and Hit-to-Lead Optimization of Non-ATP Competitive MK2 (MAPKAPK2) Inhibitors.

A novel series of non-ATP-competitive MK2 inhibitors based on a furan-2-carboxyamide scaffold was discovered through high-throughput screening using the affinity selection-mass spectrometry-based Automated Ligand Identification System platform. Medicinal chemistry efforts optimized the initial screening hit to leadlike compounds with significant improvements in biochemical and cellular potencies, while maintaining excellent kinase selectivity and in vitro pharmacokinetic properties. Biophysical and biochemical studies confirmed the unique non-ATP-competitive binding mode of this series and suggested that highly selective inhibitors of MK2 should be feasible by targeting the outside ATP pocket.

Pyridine Carboxamides: Potent Palm Site Inhibitors of HCV NS5B Polymerase.

Pyridine carboxamide-based inhibitors of the hepatitis C virus (HCV) NS5B polymerase were diversified and optimized to a variety of topologically related scaffolds. In particular, the 2-methyl nicotinic acid scaffold was developed into inhibitors with improved biochemical (IC50-GT1b = 0.014 µM) and cell-based HCV replicon potency (EC50-GT1b = 0.7 µM). Biophysical and biochemical characterization identified this novel series of compounds as palm site binders to HCV polymerase.

Discovery of cyclic acylguanidines as highly potent and selective beta-site amyloid cleaving enzyme (BACE) inhibitors: Part I--inhibitor design and validation.

A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit (3), with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores (10 and 23) were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180 degrees in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates, as revealed by 3. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.

Application of fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design to identify novel microM leads for the development of nM BACE-1 (beta-site APP cleaving enzyme 1) inhibitors.

Fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design were used to identify novel inhibitors for BACE-1. A rapid optimization of an initial NMR hit was achieved by a combination of NMR and a functional assay, resulting in the identification of an isothiourea hit with a K(d) of 15 microM for BACE-1. NMR data and the crystal structure revealed that this hit makes H-bond interactions with the two catalytic aspartates, occupies the nonprime side region of the active site of BACE-1, and extends toward the S3 subpocket (S3sp). A focused NMR-based search for heterocyclic isothiourea isosteres resulted in several distinct classes of BACE-1 active site directed compounds with improved chemical stability and physicochemical properties. The strategy for optimization of the 2-aminopyridine lead series to potent inhibitors of BACE-1 was demonstrated. The structure-based design of a cyclic acylguanidine lead series and its optimization into nanomolar BACE-1 inhibitors are the subject of the companion paper

Screening of protein kinases by ATP-STD NMR spectroscopy.

ATP-STD NMR takes advantage of Mg2+ binding to ATP to adjust the ATP affinity for protein kinases permitting a wide range of Ki's to be determined for ATP competitive ligands. Substituting Mn2+ for Mg2+ creates a paramagnetic probe (MnATP) from which the proximity of non-ATP competitive ligands can be inferred. Internal standards and references are used to reduce false positives due to protein or compound degradation. Use of the natural ATP ligand confers active site-specificity that is not available a priori from other ligand binding experiments.

Characterization of autocatalytic conversion of precursor BACE1 by heteronuclear NMR spectroscopy.

Accumulation of the cytotoxic 40- to 42-residue beta-amyloid peptide represents the primary pathological process in Alzheimer's disease (AD). BACE1 (beta-site APP cleaving enzyme 1) is responsible for the initial required step in the neuronal amyloidogenic processing of beta-amyloid precursor protein and is a major drug target for the therapeutic intervention of AD. In the present study, BACE1 is initially synthesized as an immature precursor protein containing part of the pre domain and the entire pro domain, and undergoes autocatalytic conversion to yield the well-folded mature BACE1 enzyme. To understand the mechanism of the conversion and the role of the pro domain, we monitored the autocatalytic conversion of BACE1 by heteronuclear NMR spectroscopy and used chemical shift perturbations as a probe to study the structural changes accompanying the autocatalytic conversion. NMR data revealed local conformational changes from a partially disordered to a well-folded conformation associated with the conversion. The conformational changes are largely concentrated in the NH(2)-terminal lobe. Conversely, the active site conformations are conserved during the autocatalytic conversion. The precursor and mature BACE1 proteins were further characterized for their ability to interact with a substrate-based transition state BACE1 peptide inhibitor. The precursor BACE1 rapidly adopted the bound conformation in the presence of the inhibitor, which is identical to the bound conformation of the mature protein. The interaction of the inhibitor with both the precursor BACE1 and the fully processed BACE1 is in slow exchange on the NMR time scale, indicating a tight binding interaction. Overall, the NMR data demonstrated that the pro domain does not hinder inhibitor binding and may assist in the proper folding of the protein. The fully processed BACE1 represents a high quality well-folded protein which is highly stable over a long period of time, and is suitable for evaluation of inhibitor binding by NMR for drug intervention.