Christopher A. Lepre

The SHAPES strategy: an NMR-based approach for lead generation in drug discovery.

BACKGROUNDnRecently, it has been shown that nuclear magnetic resonance (NMR) may be used to identify ligands that bind to low molecular weight protein drug targets. Recognizing the utility of NMR as a very sensitive method for detecting binding, we have focused on developing alternative approaches that are applicable to larger molecular weight drug targets and do not require isotopic labeling.nnnRESULTSnA new method for lead generation (SHAPES) is described that uses NMR to detect binding of a limited but diverse library of small molecules to a potential drug target. The compound scaffolds are derived from shapes most commonly found in known therapeutic agents. NMR detection of low (microM-mM) affinity binding is achieved using either differential line broadening or transferred NOE (nuclear Overhauser effect) NMR techniques.nnnCONCLUSIONSnThe SHAPES method for lead generation by NMR is useful for identifying potential lead classes of drugs early in a drug design program, and is easily integrated with other discovery tools such as virtual screening, high-throughput screening and combinatorial chemistry.

Crystal Structure of the Ectodomain Complex of the CGRP Receptor, a Class-B GPCR, Reveals the Site of Drug Antagonism

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

Crystal Structure of the P38α-MAPKAP Kinase 2 Heterodimer

The p38 signaling pathway is activated in response to cell stress and induces production of proinflammatory cytokines. P38α is phosphorylated and activated in response to cell stress by MKK3 and MKK6 and in turn phosphorylates a number of substrates, including MAPKAP kinase 2 (MK2). We have determined the crystal structure of the unphosphorylated p38α-MK2 heterodimer. The C-terminal regulatory domain of MK2 binds in the docking groove of p38α, and the ATP-binding sites of both kinases are at the heterodimer interface. The conformation suggests an extra mechanism in addition to the regulation of the p38α and MK2 phosphorylation states that prevents phosphorylation of substrates in the absence of cell stress. Addition of constitutively active MKK6-DD results in rapid phosphorylation of the p38α-MK2 heterodimer.

Microdrop screening: A rapid method to optimize solvent conditions for NMR spectroscopy of proteins

Determining appropriate solvent conditions is a crucial first step for carrying out NMR spectroscopy of proteins, but rapid and efficient methods for doing so are currently lacking. Microdrop screening examines a large number of different solvent conditions using very small amounts of protein and minimal labor. Starting from one initial buffer condition, small aliquots of protein solution are combined with an array of solutions in which concentration, pH, buffer type, and added stabilizers are systematically varied. The protein concentration of each microliter-sized test drop (‘microdrop’) is gradually changed using vapor diffusion, and the solubility of the protein is determined by visual examination. A variety of analytical techniques may be applied to the contents of the microdrops to monitor enzymatic activity, aggregation, ligand binding, and protein folding.

Library design for NMR-based screening.

Recent advances in NMR-based screening methods have made it possible to screen larger libraries of molecules with higher throughput. However, experience shows that intelligent library design is important if NMR screening is to succeed in aiding our discovery of potent and useful lead compounds. This review presents the current state-of-the-art methodologies for designing primary and follow-up libraries for NMR screening. Diversity, drug-likeness and combinatorial libraries are discussed, and the inherent pitfalls of the NMR approach are addressed.

Practical aspects of NMR-based fragment screening.

NMR spectroscopy is a popular and highly versatile screening method for fragment-based drug discovery. NMR methods are capable of robustly detecting the binding of fragments to macromolecular targets over an extraordinarily broad affinity range (from covalent to millimolar). This chapter provides a stepwise process for creating an NMR-based fragment screening program. The construction of fragment libraries is described, including compound selection, plating of stocks, and preparation of mixtures. Guidance is given for designing fragment screens, such as choosing the appropriate NMR screening format and method, and optimizing the sample conditions and experimental parameters. The identification and validation of screening hits is described, and a number of potential pitfalls are discussed. Rather than detailing one specific screening protocol, this chapter outlines the available options and provides information to enable users to design their own customized fragment screening programs.

Leveraging structural approaches: applications of NMR-based screening and X-ray crystallography for inhibitor design.

In the last several years, NMR strategies in drug discovery have evolved from a primarily structural focus to a set of technologies that are non-structural in nature but that have a much greater impact on the identification and optimization of real drug leads. NMR-based screening methods, such as the SHAPES strategy, help rapidly identify good starting points for drug design in a relatively high throughput implementation. The SHAPES method uses simple NMR techniques to detect binding of a limited, but diverse library of low molecular weight, soluble compounds to a potential drug target. SHAPES library compounds are derived largely from molecular frameworks most commonly found in known therapeutic agents. The NMR experiments used in these protocols are based on the well-known NMR techniques, and may be applied to targets with no limitation on molecular weight and no requirement for isotope labeling. Following screening, SHAPES hits may be used to guide virtual screening, synthesis of combinatorial libraries, and bias the first compounds that undergo high throughput screening. Integration of the SHAPES strategy with iterative X-ray crystallographic structure determination can be very useful in deriving an initial structural pharmacophore model and achieving significant in vitro potency in a short time frame. Here, examples are provided of how the combination of NMR SHAPES screening, virtual screening, molecular modeling and X-ray crystallography has led to novel drug scaffolds in several drug discovery programs: JNK3 MAP kinase and the fatty acid binding protein, aP2.

Solution structure of FK506 bound to FKBP‐12

The complex of the immunosuppressant FK506 bound to FKBP‐12 has been studied in solution using 1H and inverse‐detected 13C NMR methods. The resonances of bound, 13C‐labelled FK506 were assigned and a set of 66 intraligand NOE distance restraints were used to calculate the structure of the bound ligand by distance geometry and restrained molecular dynamics methods. The structure of bound FK506 in solution closely resembles that seen in the X‐ray structure [17], except for the allyl region. The differences reflect the influence of intermolecular crystal contacts and have implications for interpretation of the interaction of the FK506/FKBP complex with its putative biological receptor.

Fragment-based drug discovery using the SHAPES method

The SHAPES method is one of several fragment-based drug discovery methods developed in the last decade. Molecules containing drug-like fragments are screened using NMR methods to find weakly binding (0.1 μM to multi-mM) hits that are then transformed into potent, viable leads. This review analyzes ten years of SHAPES screens, in which potent leads were found for 70 – 80% of the targets screened, and discusses lessons learned about how best to apply fragment-based lead discovery in the pharmaceutical environment. Detailed examples of lead discovery and optimization for the kinases REDK and MK2 are given. Finally, future directions are considered and a strategy is proposed for increasing efficiency by coupling fragment-based screening with receptor-assisted inhibitor design (NMR-RAID).

Fragment-Based Discovery of Dual JC Virus and BK Virus Helicase Inhibitors

There are currently no treatments for life-threatening infections caused by human polyomaviruses JCV and BKV. We therefore report herein the first crystal structure of the hexameric helicase of JCV large T antigen (apo) and its use to drive the structure-based design of dual JCV and BKV ATP-competitive inhibitors. The crystal structures obtained by soaking our early inhibitors into the JCV helicase allowed us to rapidly improve the biochemical activity of our inhibitors from 18 μM for the early 6-(2-methoxyphenyl)- and the 6-(2-ethoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole hits 1a and 1b to 0.6 μM for triazolopyridine 12i. In addition, we were able to demonstrate measurable antiviral activity in Vero cells for our thiazolopyridine series in the absence of marked cytotoxicity, thus confirming the usefulness of this approach.