Navigating the kinome.

Although it is increasingly being recognized that drug-target interaction networks can be powerful tools for the interrogation of systems biology and the rational design of multitargeted drugs, there is no generalized, statistically validated approach to harmonizing sequence-dependent and pharmacology-dependent networks. Here we demonstrate the creation of a comprehensive kinome interaction network based not only on sequence comparisons but also on multiple pharmacology parameters derived from activity profiling data. The framework described for statistical interpretation of these network connections also enables rigorous investigation of chemotype-specific interaction networks, which is critical for multitargeted drug design.

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.

An inhibitor of Bcl-2 family proteins induces regression of solid tumours.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

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.

Biochemical and biophysical characterization of unique switch pocket inhibitors of p38α.

Herein we describe the identification and characterization of a class of molecules that are believed to extend into a region of p38 known as the 'switch pocket'. Although these molecules lack a canonical hinge binding motif, they show K(i) values as low as 100 nM against p38. We show that molecules that interact with this region of the protein demonstrate different binding kinetics than a canonical ATP mimetic, as well as a wide range of kinome profiles. Thus, the switch pocket presents new opportunities for kinome selectivity which could result in unique biochemical responses and offer new opportunities in the field of kinase drug discovery.

Discovery of a potent and selective Bcl-2 inhibitor using SAR by NMR.

The Bcl-2 family of proteins plays a major role in the regulation of apoptosis, or programmed cell death. Overexpression of the anti-apoptotic members of this family (Bcl-2, Bcl-x(L), and Mcl-1) can render cancer cells resistant to chemotherapeutic agents and therefore these proteins are important targets for the development of new anti-cancer agents. Here we describe the discovery of a potent, highly selective, Bcl-2 inhibitor using SAR by NMR and structure-based drug design which could serve as a starting point for the development of a Bcl-2 selective anti-cancer agent. Such an agent would potentially overcome the Bcl-x(L) mediated thrombocytopenia observed with ABT-263.

A decade of fragment-based drug design: strategic advances and lessons learned.

Since the early 1990s, several technological and scientific advances - such as combinatorial chemistry, high-throughput screening and the sequencing of the human genome - have been heralded as remedies to the problems facing the pharmaceutical industry. The use of these technologies in some form is now well established at most pharmaceutical companies; however, the return on investment in terms of marketed products has not met expectations. Fragment-based drug design is another tool for drug discovery that has emerged in the past decade. Here, we describe the development and evolution of fragment-based drug design, analyse the role that this approach can have in combination with other discovery technologies and highlight the impact that fragment-based methods have made in progressing new medicines into the clinic.

Molecular determinants of species-specific activation or blockade of TRPA1 channels.

TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal). However, how covalent modification leads to channel opening is not understood. Here, we report that electrophilic, thioaminal-containing compounds [e.g., CMP1 (4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide)] covalently modify cysteine residues but produce striking species-specific effects [i.e., activation of rat TRPA1 (rTRPA1) and blockade of human TRPA1 (hTRPA1) activation by reactive and nonreactive agonists]. Through characterizing rTRPA1 and hTRPA1 chimeric channels and point mutations, we identified several residues in the upper portion of the S6 transmembrane domains as critical determinants of the opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, whereas equivalent residues of hTRPA1 (Ser-943 and Ile-946) determine channel block. Furthermore, side-chain replacements at these critical residues profoundly affect channel function. Therefore, our findings reveal a molecular basis of species-specific channel gating and provide novel insights into how TRPA1 respond to stimuli.

Structural characterization of a soluble amyloid beta-peptide oligomer.

Alzheimer's disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid beta-peptides that can form insoluble fibrils or soluble oligomeric assemblies. Soluble forms are present in the brains and tissues of Alzheimer's patients, and their presence correlates with disease progression. Long-lived soluble forms can be generated in vitro by using small amounts of aliphatic hydrocarbon chains of detergents or fatty acids in preparations of amyloid beta-peptides. Using NMR, we have characterized soluble oligomers of Abeta preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel beta-sheet structure that is different from fibrils which contain only parallel beta-sheets. Using the structural data, we engineered a disulfide bond into the soluble Abeta globulomer to give a "new" soluble antigen that is stable, homogeneous, and binds with the same affinity to selective antibodies as the parent wt globulomer.

Statistical analysis of the effects of common chemical substituents on ligand potency.

The results of a statistical analysis of more than 84,000 compounds from lead optimization programs against 30 different protein targets is presented, with a focus on the effects that different chemical substituents have on compound potency. It is observed that the potency changes induced by most chemical groups follows a nearly normal distribution centered near zero (i.e., no effect on potency). However, the widths of the distributions vary significantly between different substituents, and these effects cannot be rationalized by simple physicochemical parameters. In addition, certain substituents consistently bias the distribution toward higher or lower potency, suggesting the existence of preferred and nonpreferred chemical groups for lead optimization. The implications of these results for understanding protein-ligand recognition and for enhancing the efficiency and speed of lead optimization will be discussed.

Scaffold oriented synthesis. Part 2: Design, synthesis and biological evaluation of pyrimido-diazepines as receptor tyrosine kinase inhibitors.

We report the discovery of the pyrimido-diazepine scaffolds as novel adenine mimics. Structure-based design led to the discovery of analogs with potent inhibitory activity against receptor tyrosine kinases, such as KDR, Flt3 and c-Kit. Compound 14 exhibited low nanomolar KDR enzymatic and cellular potencies (IC(50)=9 and 52 nM, respectively).

Solution structure and calcium-binding properties of EF-hands 3 and 4 of calsenilin.

Calsenilin is a member of the recoverin branch of the EF-hand superfamily that is reported to interact with presenilins, regulate prodynorphin gene expression, modulate voltage-gated Kv4 potassium channel function, and bind to neurotoxins. Calsenilin is a Ca+2-binding protein and plays an important role in calcium signaling. Despite its importance in numerous neurological functions, the structure of this protein has not been reported. In the absence of Ca+2, the protein has limited spectral resolution that increases upon the addition of Ca+2. Here, we describe the three-dimensional solution structure of EF-hands 3 and 4 of calsenilin in the Ca+2-bound form. The Ca+2-bound structure consists of five alpha-helices and one two-stranded antiparallel beta-sheet. The long loop that connects EF hands 3 and 4 is highly disordered in solution. In addition to its structural effects, Ca+2 binding also increases the protein's propensity to dimerize. These changes in structure and oligomerization state induced upon Ca+2 binding may play important roles in molecular recognition during calcium signaling.

Nuclear magnetic resonance in target profiling and compound file enhancement.

Nuclear magnetic resonance (NMR) has matured as an important tool in drug discovery and development, with firm establishment of its roles in lead generation and optimization through application of NMR-based fragment screening and structure-based drug design. Besides these applications, NMR technology has expanded to make contributions both earlier and later in the drug discovery process. This review will focus on the impact of NMR in the early stages of drug discovery, in particular in characterizing the viability of targets for further discovery exercises and improving high-throughput screening through compound file-enhancement initiatives.

Fragment-based drug design: how big is too big?

Much has been discussed about the proper physicochemical properties (e.g., molecular weight, hydrophobicity, etc.) that should be considered when utilizing fragment leads in drug design. However, little has been reported as to what emphasis, if any, should be placed on the potency of the resulting fragment leads. In this report, a retrospective analysis of 18 highly optimized inhibitors is described in which the compounds were systematically deconstructed until the minimal binding elements could be identified. An analysis of the potency changes that were observed as the leads were reduced in size indicate that a nearly linear relationship exists between molecular weight and binding affinity over the entire range of sizes and potencies represented in the dataset. On the basis of these observations, prediction maps can be constructed that enable critical and quantitative assessments of the process of lead identification and optimization. These data place well-defined limits on the ideal size and potency of fragment leads that are being considered for use in fragment-based drug design.

Discovery of a potent inhibitor of the antiapoptotic protein Bcl-xL from NMR and parallel synthesis.

The antiapoptotic proteins Bcl-x(L) and Bcl-2 play key roles in the maintenance of normal cellular homeostasis. However, their overexpression can lead to oncogenic transformation and is responsible for drug resistance in certain types of cancer. This makes Bcl-x(L) and Bcl-2 attractive targets for the development of potential anticancer agents. Here we describe the structure-based discovery of a potent Bcl-x(L) inhibitor directed at a hydrophobic groove on the surface of the protein. This groove represents the binding site for BH3 peptides from proapoptotic Bcl-2 family members such as Bak and Bad. Application of NMR-based screening yielded an initial biaryl acid with an affinity (K(d)) of approximately 300 microM for the protein. Following the classical "SAR by NMR" approach, a second-site ligand was identified that bound proximal to the first-site ligand in the hydrophobic groove. From NMR-based structural studies and parallel synthesis, a potent ligand was obtained, which binds to Bcl-x(L) with an inhibition constant (K(i)) of 36 +/- 2 nM.

TINS, target immobilized NMR screening: an efficient and sensitive method for ligand discovery.

We propose a ligand screening method, called TINS (target immobilized NMR screening), which reduces the amount of target required for the fragment-based approach to drug discovery. Binding is detected by comparing 1D NMR spectra of compound mixtures in the presence of a target immobilized on a solid support to a control sample. The method has been validated by the detection of a variety of ligands for protein and nucleic acid targets (K(D) from 60 to 5000 muM). The ligand binding capacity of a protein was undiminished after 2000 different compounds had been applied, indicating the potential to apply the assay for screening typical fragment libraries. TINS can be used in competition mode, allowing rapid characterization of the ligand binding site. TINS may allow screening of targets that are difficult to produce or that are insoluble, such as membrane proteins.

Structure of MurF from Streptococcus pneumoniae co-crystallized with a small molecule inhibitor exhibits interdomain closure.

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.

Predicting protein druggability.

The ability to predict whether a particular protein can bind with high affinity and specificity to small, drug-like compounds based solely on its 3D structure has been a longstanding goal of structural biologists and computational scientists. The promise is that an accurate prediction of protein druggability can capitalize on the huge investments already made in structural genomics initiatives by identifying highly druggable proteins and using this information in target identification and validation campaigns. Here we discuss the potential utility of tools that characterize protein targets and describe strategies for the optimal integration of protein druggability data with bioinformatic approaches to target selection.

Utilization of NMR-derived fragment leads in drug design.

The advent of large-scale NMR-based screening has enabled new strategies for the design of novel, potent inhibitors of therapeutic targets. In particular, fragment-based strategies, in which molecular portions of the final high-affinity ligand are experimentally identified prior to chemical synthesis, have found widespread utility. This chapter will discuss some of the practical considerations for identifying and utilizing these fragment leads in drug design, with special emphasis on some of the lessons learned from more than a decade of industry experience.