Perspectives on Nuclear Magnetic Resonance Spectroscopy in Drug Discovery Research.

The drug discovery landscape has undergone a significant transformation over the past decade, owing to research endeavors in a wide range of areas leading to strategies for pursuing new drug targets and the emergence of novel drug modalities. NMR spectroscopy has been a technology of fundamental importance to these research pursuits and has seen its use expanded both within and outside of traditional medicinal chemistry applications. In this perspective, we will present advancement of NMR-derived methods that have facilitated the characterization of small molecules and novel drug modalities including macrocyclic peptides, cyclic dinucleotides, and ligands for protein degradation. We will discuss innovations in NMR spectroscopy at the chemistry and biology interface that have broadened NMR's utility from hit identification through lead optimization activities. We will also discuss the promise of emerging NMR approaches in bridging our understanding and addressing challenges in the pursuit of the therapeutic agents of the future.

Complex peptide macrocycle optimization: combining NMR restraints with conformational analysis to guide structure-based and ligand-based design.

Systematic optimization of large macrocyclic peptide ligands is a serious challenge. Here, we describe an approach for lead-optimization using the PD-1/PD-L1 system as a retrospective example of moving from initial lead compound to clinical candidate. We show how conformational restraints can be derived by exploiting NMR data to identify low-energy solution ensembles of a lead compound. Such restraints can be used to focus conformational search for analogs in order to accurately predict bound ligand poses through molecular docking and thereby estimate ligand strain and protein-ligand intermolecular binding energy. We also describe an analogous ligand-based approach that employs molecular similarity optimization to predict bound poses. Both approaches are shown to be effective for prioritizing lead-compound analogs. Surprisingly, relatively small ligand modifications, which may have minimal effects on predicted bound pose or intermolecular interactions, often lead to large changes in estimated strain that have dominating effects on overall binding energy estimates. Effective macrocyclic conformational search is crucial, whether in the context of NMR-based restraints, X-ray ligand refinement, partial torsional restraint for docking/ligand-similarity calculations or agnostic search for nominal global minima. Lead optimization for peptidic macrocycles can be made more productive using a multi-disciplinary approach that combines biophysical data with practical and efficient computational methods.

Ranking mAb-excipient interactions in biologics formulations by NMR spectroscopy and computational approaches.

Excipients are added to biopharmaceutical formulations to enhance protein stability and enable the development of robust formulations with acceptable physicochemical properties, but the mechanism by which they confer stability is not fully understood. Here, we aimed to elucidate the mechanism through direct experimental evidence of the binding affinity of an excipient to a monoclonal antibody (mAb), using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method. We ranked a series of excipients with respect to their dissociation constant (KD) and nonspecific binding constants (Ns). In parallel, molecular dynamic and site identification by ligand competitive saturation (SILCS)-Monte Carlo simulations were done to rank the excipient proximity to the proteins, thereby corroborating the ranking by STD NMR. Finally, the excipient ranking by NMR was correlated with mAb conformational and colloidal stability. Our approach can aid excipient selection in biologic formulations by providing insights into mAb-excipient affinities before conventional and time-consuming excipient screening studies are conducted.

An improved protocol for amino acid type-selective isotope labeling in insect cells.

An improved expression protocol is proposed for amino acid type-specific [13C], [15N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449-456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents.

NMR in drug design.

The use of NMR as a tool to determine 3 dimensional protein solution structures, once a darling of the pharmaceutical industry, has largely given way to study of the interaction of prospective drugs with macromolecular targets. Many of these approaches involve ligand-centered studies, which have the advantage of speed and efficiency, but there are also many approaches that take directly from our learnings in macromolecular NMR and provide greater structural detail yet are still optimized for rapid turn-around of information. In the evolution of NMR in the pharmaceutical industry, the unique strengths of NMR to provide dynamic and atomic level information continue to be exploited to discover and design new drugs. Numerous methods have been developed over the past two decades that fall into the categories of fragment-based pre-lead discovery, ligand binding studies and qualitative structural screening.

Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis.

Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here, we report the identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics. Five peptide regions on IL-23 with reduced backbone amide solvent accessibility upon antibody binding were identified by HDX-MS, and five different peptides over the same three regions were identified by FPOP. In addition, FPOP analysis at the residue level reveals potentially key interacting residues. Mutants with 3-5 residues changed to alanine have no measurable differences from wild-type IL-23 except for binding of and signaling blockade by the 7B7 anti-IL-23 antibody. The M5 IL-23 mutant differs from wild-type by five alanine substitutions and represents the dominant energetic epitope of 7B7. M5 shows a dramatic decrease in binding to BMS-986010 (which contains the 7B7 Fab, where Fab is fragment antigen-binding region of an antibody), yet it maintains functional activity, binding to p40 and p19 specific reagents, and maintains biophysical properties similar to wild-type IL-23 (monomeric state, thermal stability, and secondary structural features).

Discovery of a Potent Acyclic, Tripeptidic, Acyl Sulfonamide Inhibitor of Hepatitis C Virus NS3 Protease as a Back-up to Asunaprevir with the Potential for Once-Daily Dosing.

The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure-activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).

Atropisomer Control in Macrocyclic Factor VIIa Inhibitors.

Incorporation of a methyl group onto a macrocyclic FVIIa inhibitor improves potency 10-fold but is accompanied by atropisomerism due to restricted bond rotation in the macrocyclic structure, as demonstrated by NMR studies. We designed a conformational constraint favoring the desired atropisomer in which this methyl group interacts with the S2 pocket of FVIIa. A macrocyclic inhibitor incorporating this constraint was prepared and demonstrated by NMR to reside predominantly in the desired conformation. This modification improved potency 180-fold relative to the unsubstituted, racemic macrocycle and improved selectivity. An X-ray crystal structure of a closely related analogue in the FVIIa active site was obtained and matches the NMR and modeled conformations, confirming that this conformational constraint does indeed direct the methyl group into the S2 pocket as designed. The resulting rationally designed, conformationally stable template enables further optimization of these macrocyclic inhibitors.

Discovery of novel P1 groups for coagulation factor VIIa inhibition using fragment-based screening.

A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles. Crystallographic structures for several bound fragments were obtained, leading to the successful design of a potent factor VIIa inhibitor with a neutral lactam P1 and improved permeability.

The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).

Design, synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: observation of extensive dynamics in an enzyme/inhibitor complex.

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH2 group, inhibits HDHD4 with a binding affinity (IC50 11µM) in the range of the native substrate Neu5Ac-9-P (compound 1, Km 47µM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg(2+). In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca(2+) and Mg(2+) are indicative of a highly dynamic process in the active site for the HDHD4/Mg(2+)/3 complex. Possible explanations for this observation are discussed.

The dynamic process of ß(2)-adrenergic receptor activation.

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ß(2)-adrenergic receptor (ß(2)AR), a prototypical GPCR. We labeled ß(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ß(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ß(2)AR's ability to engage multiple signaling and regulatory proteins.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

Multiple and single binding modes of fragment-like kinase inhibitors revealed by molecular modeling, residue type-selective protonation, and nuclear overhauser effects.

Fragment-like inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK2) include 5-hydroxyisoquinoline (IC50 approximately 85 microM). Modeling studies identified four possible binding modes for this compound. Two-dimensional (1)H-(1)H NOESY data obtained with selectively protonated samples of MK2 in complex with 5-hydroxyisoquinoline demonstrated that two of the four predicted binding modes are well populated. A second small isoquinoline was subsequently shown to bind in a single mode. NMR and modeling studies using this general approach are expected to facilitate "scaffold hopping" and structure-guided elaborations of fragment-like kinase inhibitor cores.

Alternate HMQC experiments for recording HN and HC-correlation spectra in proteins at high throughput.

Alternate implementations of the SOFAST-HMQC experiment are described. In these alternate SOFAST-HMQC experiments (ALSOFAST-HMQC) excitation of the magnetization of interest is achieved by non-selective rf pulses while preserving the equilibrium polarization of passive spins. This alternate excitation scheme also allows the incorporation of a novel sensitivity enhancement protocol which has been most recently developed by Brutscher and coworkers and which permits simultaneous detection of both the x- and y-components of the indirectly detected t(1)-interferograms without the need to introduce additional rf pulses and delays. We show that the ALSOFAST HC-HMQC experiment, which implements an alternate means of frequency selection, enables the detection of methyl resonances with large secondary proton chemical shifts. This is achieved by selecting coherences of interest via a frequency selective carbon inversion pulse. Detailed comparisons between SOFAST- and the presented ALSOFAST-HMQC experiment reveals a considerable degree of mutual complementarity.

NMR structure of a potent small molecule inhibitor bound to human keratinocyte fatty acid-binding protein.

The NMR structure is presented for compound 1 (BMS-480404) (Ki = 33 (+/-2) nM) bound to keratinocyte fatty acid-binding protein. This article describes interactions between a high affinity drug-like compound and a member of the fatty acid-binding protein family. A benzyl group ortho to the mandelic acid in 1 occupies an area of the protein that fatty acids do not normally contact. Similar to that in the kFABP-palmitic acid structure, the acid moiety in 1 is proximal to R129 and Y131. Computational modeling indicates that the acid moiety in 1 interacts indirectly via a modeled water molecule to R109.

Metabolomic analysis using optimized NMR and statistical methods.

NMR-based metabolomics requires robust automated methodologies, and the accuracy of NMR-based metabolomics data is greatly influenced by the reproducibility of data acquisition and processing methods. Effective water resonance signal suppression and reproducible spectral phasing and baseline traces across series of related samples are crucial for statistical analysis. We assess robustness, repeatability, sensitivity, selectivity, and practicality of commonly used solvent peak suppression methods in the NMR analysis of biofluids with respect to the automated processing of the NMR spectra and the impact of pulse sequence and data processing methods on the sensitivity of pattern recognition and statistical analysis of the metabolite profiles. We introduce two modifications to the excitation sculpting pulse sequence whereby the excitation solvent suppression pulse cascade is preceded by low-power water resonance presaturation pulses during the relaxation delay. Our analysis indicates that combining water presaturation with excitation sculpting water suppression delivers the most reproducible and information-rich NMR spectra of biofluids.

Protein-ligand NOE matching: a high-throughput method for binding pose evaluation that does not require protein NMR resonance assignments.

Given the three-dimensional (3D) structure of a protein, the binding pose of a ligand can be determined using distance restraints derived from assigned intra-ligand and protein-ligand nuclear Overhauser effects (NOEs). A primary limitation of this approach is the need for resonance assignments of the ligand-bound protein. We have developed an approach that utilizes data from 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectra for evaluating ligand binding poses without requiring protein NMR resonance assignments. Only the 1H NMR assignments of the bound ligand are essential. Trial ligand binding poses are generated by any suitable method (e.g., computational docking). For each trial binding pose, the 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectrum is predicted, and the predicted and observed patterns of protein-ligand NOEs are matched and scored using a fast, deterministic bipartite graph matching algorithm. The best scoring (lowest "cost") poses are identified. Our method can incorporate any explicit restraints or protein assignment data that are available, and many extensions of the basic procedure are feasible. Only a single sample is required, and the method can be applied to both slowly and rapidly exchanging ligands. The method was applied to three test cases: one complex involving muscle fatty acid-binding protein (mFABP) and two complexes involving the leukocyte function-associated antigen 1 (LFA-1) I-domain. Without using experimental protein NMR assignments, the method identified the known binding poses with good accuracy. The addition of experimental protein NMR assignments improves the results. Our "NOE matching" approach is expected to be widely applicable; i.e., it does not appear to depend on a fortuitous distribution of binding pocket residues.