Genetic screens have long been used as an approach to identify and validate new targets for drug discovery. The vast majority of these have been carried out in cell lines: mostly cancer cell lines. However, with improvements in tissue culture techniques, the increasing interest in using the immune system to tackle disease and the discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 mediated genome editing, screening primary cells that have not been subverted by transformation into immortal lines, is both appealing and feasible. We have successfully carried out a CRISPR-Cas9 screen in primary T cells using a combined lentivirus and electroporation protocol. Freshly isolated primary T cells are stimulated with anti-CD3 and anti-CD28 antibodies and then transduced with a pooled sgRNA library. After antibiotic selection, T cells successfully transduced with sgRNAs are electroporated to introduce Cas9 mRNA. We chose to validate this approach by carrying out a screen similar to that published by Birsoy et al., 2015, in which they ran a CRISPR-Cas9 screen in Jurkat T cells in the presence and absence of the electron transport chain inhibitor phenformin. In our screen, we exposed the CRISPR-Cas9 edited pool of primary T cells to a dose of phenformin that resulted in growth inhibition to a similar degree to that used by Birsoy and colleagues. Our data are in agreement with the published screen, showing that loss of the cytosolic aspartate aminotransferase GOT1 sensitises primary T cells to phenformin. We took multiple time points in our primary T cell screen and used T cells isolated from three different donors, allowing for the analysis of guide drop-out kinetics and reproducibility between donors. We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells (MDSCs). With a view to this, we have also carried out an arrayed siRNA screen in MDSCs to look for genes that when knocked down reduce the capacity of MDSCs to inhibit T cell proliferation. The endpoint for this screen is based on co-culture of siRNA transfected MDSCs with proliferating primary T cells. Using this complex data set, we have identified several potential targets, which when validated could provide new therapeutic targets through which the immunosuppressive nature of MDSCs in the tumour microenvironment can be mitigated. Birsoy, K., et al. (2015) http://dx.doi.org/10.1016/j.cell.2015.07.016 Citation Format: Bronwyn Joubert, Cristina Ghirelli, Isabelle Nett, John Prime, Glynn Martin, Jonathan Moore, Benedict Cross, Nicola J. McCarthy. RNA-based screens in primary human immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1213.

A major focus in immuno-oncology research is finding new immuno-oncology targets, including those that alter the character and frequency of T-cell-mediated anti-tumour responses. Screens using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9-mediated genome editing seem well placed to identify new targets. However, although CRISPR-Cas9 gene editing works well in primary T cells using electroporation, use of a lentivirus one vector system has proved challenging in primary T cells compared with cancer cell lines. We have used several different approaches to identify the most useful method for transduction of primary human T cells with CRISPR components. Electroporation of sgRNAs and mRNA encoding Cas9 into proliferating T cells efficiently generate T cells with specific gene knock-outs or knock-ins, with targeting rates of around 37% for gene knockout. Thus, primary T cells are amenable to CRISPR-Cas9 gene editing, and the capacity to rapidly modify loci enables generation of primary T cell models suitable for comprehending the function of modified receptor-ligand pairs involved in an immune checkpoint response. Our pooled sgRNA-Cas9 screens in cancer cell lines have used our in-house sgRNA libraries, which include a modified tracrRNA component improving Cas9 affinity and subsequently the performance of a typical sgRNA for promoting gene editing. However, use of the same approach in primary T cells has not resulted in efficient transduction of the library. Specifically, isolated CD3+ T cells stimulated in vitro with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2 resulted in no expression or low level expression of GFP after cells were transduced with a one vector CRISPR-Cas9 sgRNA library. Our experiments indicate, in line with published data, that T cells can be transduced effectively with lentivirus, thus we are examining the use of a two vector CRISPR-Cas9 system and the use of CRISPRi to idealise CRISPR screening in primary T cells. We are also carrying out target identification and validation in myeloid derived suppressor cells (MDSCs). We are using an siRNA approach in these cells, which are generated by PBMC co-culture with cancer cell lines for 7 days, or by culture in the presence of recombinant GM-CSF and IL-6 for 7 days. Our initial data indicate that these MDSCs can effectively suppress autologous, as well as allogeneic, CD8 T cell proliferation mediated by anti-CD3 and anti-CD28 stimulation and that siRNA knockdown is effective in MDSCs. We will use our druggable genome plus arrayed siRNA library to identify targets that when knocked down inhibit the capacity of MDSCs to suppress T cell proliferation. We anticipate that these data will be useful in identifying new targets that are involved in regulating an immune response to tumour development and progression. Citation Format: Cristina Ghirelli, Thibault Laurent, Simon Scrace, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore. CRISPR-Cas9 and siRNA screening in primary human immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4123. doi:10.1158/1538-7445.AM2017-4123

Abstract Horizon is establishing a powerful in vitro immuno-oncology platform with the capability to identify combinations of agents that will synergise with checkpoint inhibitors, determine methods for making safer and more effective cell therapies and find novel targets for immuno-therapy. Horizon previously reported the establishment of a platform consisting of a suite of high-throughput cell-based assays modelling a variety of oncology relevant immune reactions. The platform includes primary human immune-cell based assays for T cell activation, Mixed lymphocyte reaction (MLR), Tumor cell lysis, Antibody-dependent cellular cytotoxicity (ADCC), Complement-dependent cytotoxicity (CDC) and Natural killer cell cytotoxicity assays that have been validated with appropriate clinically approved antibodies (e.g. nivolumab, blinotumamab, & rituximab). Miniaturized to a 384-well format and supported by automation at each experimental step the platform is highly customizable in terms of testing agents that either enhance or inhibit immune cell functions. We will present ongoing efforts to identify synergistic activities with other relevant therapeutics likely to bring benefits to patients. In addition to the continued development of our high throughput immuno-oncology cell based assays, we have expanded our cell engineering know how combined with CRISPR-Cas9 gene editing technology to build new models to understand better the immune response and its subversion in tumorigenesis. By modifying specific genes of interest in immune cells we aim to robustly identify and validate new targets for the clinic. For example, we have used high efficiency gene editing with CRISPR-Cas9 in human primary T cells to knock out and knock in genes using Neon transfection systems. Using this methodology we can rapidly generate primary T cell models lacking specific checkpoint proteins (such as PD-1) to better understand how T-cell signalling pathways interact and to nominate novel targets suitable for ex vivo gene editing. We have also employed pooled CRISPR-Cas9 screens to investigate the effects of metabolic changes on T cell biology. CD3+ T cells have been infected with a one vector lentiviral system that delivers both Cas9 and sgRNAs targeting genes involved in cellular metabolism. Results from these screens should identify potential targets involved in T cell metabolism that affect their capacity to respond to proliferative stimuli in the form of anti-CD3 and anti-CD28 antibodies. We anticipate that these and other data generated using libraries targeting essential genes will be invaluable for the design of more complex immuno-oncology screens. Overall, Horizon's integrated immuno-oncology platform will enable large scale interrogation of prospective immunotherapies, either alone or in combination, and could be useful for the discovery of novel checkpoint components, for deciphering the underlying mechanisms promoting an immunosuppressive tumor microenvironment and for the understanding of how pathways crucial for an anti-tumor immune response interact. Thus, this platform could contribute substantially to the discovery and development of future immunotherapies. Citation Format: An Frank, Sujatha Kumar, Christina Ghirelli, Kim Hoenderdos, Tabasum Huseni, Lauren Thibault, Lydia Kifle, Nava Almog, Felicia Zhao, Simon Scrace, Anatoly Myaskovsky, Chris Lowe, Janine Steiger, Nicola McCarthy, Jonathan Moore. An integrated immuno-oncology platform using high-throughput cell based assays, gene editing and genomic screens in immune cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B66.

Nanodiscs and isotropic bicelles are promising membrane mimetics in the field of solution nuclear magnetic resonance (NMR) spectroscopy of integral membrane proteins (IMPs). Despite varied challenges to solution NMR studies of IMPs, we attribute the paucity of solution NMR structures in these environments to the inability of diverse IMPs to withstand detergent treatment during standard nanodisc and bicelle preparations. Here, we present a strategy that creates small isotropic bicelles from IMPs co-translationally embedded in large nanodiscs using cell-free expression. Our results demonstrate appreciable gains in NMR spectral quality while preserving lipid-IMP contacts. We validate the approach on the detergent-sensitive LspA, which finally allowed us to perform high-quality triple-resonance NMR experiments for structural studies. Our strategy of producing bicelles from nanodiscs comprehensively avoids detergent during expression and preparation and is suitable for solution NMR spectroscopy of lipid-IMP complexes.

, Summary Nanodiscs and isotropic bicelles are promising membrane mimetics in the field of solution NMR spectroscopy of integral membrane proteins (IMPs). Despite varied challenges to solution NMR studies of IMPs, we attribute the paucity of solution NMR structures in these environments to the inability of diverse IMPs to withstand detergent treatment during standard nanodisc and bicelle preparations. Here, we present a strategy that creates small isotropic bicelles from IMPs cotranslationally embedded in large nanodiscs using cell-free expression. Our results demonstrate appreciable gains in NMR spectral quality while preserving lipid-IMP contacts. We validate the approach on the detergent sensitive LspA, which finally allowed us to perform high quality triple resonance NMR experiments for structural studies. Our strategy of producing bicelles from nanodiscs comprehensively avoids detergent during expression and preparation and is suitable for solution NMR spectroscopy of lipid-IMPs complexes. Graphical abstract Laguerre et al. show that nanodisc bilayers can be peeled away from embedded membrane proteins by detergent titration to make bicelles. Avoiding initial detergent solubilization, this method preserves lipid contacts and functional folds of detergent-sensitive membrane proteins. The resulting improvements in spectral intensity facilitate high resolution NMR spectroscopy for structure determination.

Recent clinical data indicate immunotherapy can be an effective treatment for cancer patients, yielding dramatically increased survival times in some cases. As not all patients benefit from treatments such as anti-CTLA4 and anti-PD1 antibodies, a major focus in immuno-oncology research is finding new immuno-oncology targets, including those that alter the character and frequency of T-cell-mediated anti-tumor responses. We have had great success using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 mediated genome editing to probe gene function in cancer cells via the generation of knock-out and knock-in mutants. We are now applying this approach to primary immune cells and are deploying both CRISPR–Cas9 cell engineering and CRISPR–Cas9 screens to better understand T cell biology and to find new therapeutic targets. We use T cells negatively purified by magnetic sorting from peripheral blood mononuclear cells taken from healthy donors. Our initial work in primary human T cells has focused on the capacity to knock out and knock in genes using the Neon™ transfection system. Our data indicate that primary T cells are amenable to gene editing, and the capacity to rapidly modify loci, such as PDCD1, which encodes PD-1 enables generation of primary T cell models suitable for comprehending the function of modified receptor–ligand pairs involved in an immune checkpoint response. Our pooled sgRNA–Cas9 screens have used our in-house sgRNA libraries, which include a modified tracrRNA component improving Cas9 affinity and subsequently the performance of a typical sgRNA for promoting gene editing and modifying phenotype. Briefly, isolated CD3+ T cells are stimulated in vitro with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2. Next, the proliferating T cells are co-cultured with a GFP expressing Lentivirus that directs expression of Cas9 and an sgRNA drawn from a 3900 member sgRNA library targeting genes involved in the regulation of metabolism and a control library of 2442 guide RNAs. After prolonged Lentivirus and T cell co-culture, transduced T cells are sorted based on their GFP expression and periodically re-stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 to allow their proliferation and expansion over several weeks. Representative cell pellets are taken after GFP sorting and then at specific time points throughout the screen. For each time point, gDNA is extracted and PCR is carried out to isolate the gRNAs that are present in each cell and these are analysed using NGS. NGS results are interpreted using algorithms from the previously published model-based analysis of genome-wide CRISPR–Cas9 knockout (MAGeCK) and Bayesian normalisation of gene expression levels (BAGEL) approaches. These screens are currently ongoing and are being run in T cells isolated from five independent donors to assess the impact of donor variability. We will present results from these screens assessing guide drop-out kinetics and reproducibility by comparing the performance of specific guides over multiple time points in each of the donors. We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells, involved in regulating the immune response to tumor development and progression. Citation Format: Cristina Ghirelli, Thibault Laurent, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore. CRISPR-Cas9 engineering and screening in primary human T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B086.

Calcitonin gene-related peptide (CGRP) has been implicated in acute migraine pathogenesis. In an effort to identify novel CGRP receptor antagonists for the treatment of migraine, we have discovered thiazolidinone 49, a potent (Ki=30pM, IC50=1nM), orally bioavailable, CNS-penetrant CGRP antagonist with good pharmacokinetic properties.

Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of C-13 90 degrees pulses allows to either select or discriminate against C-13' or 13C(alpha) spins coupled to N-15. As a result, H-1-N-15 correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns. (C) 2014 Elsevier Inc. All rights reserved.

Abstract Pyruvate dehydrogenase kinase (PDK) regulates the activity of the pyruvate dehydrogenase complex (PDC) through phosphorylation of three serine residues on the E1α subunit, resulting in decreased activity. The expression of all four mammalian isoforms of PDK have been shown to be up-regulated either under tumour relevant conditions (PDK-1 & PDK-3 by hypoxia) or by the loss of function of common tumour suppressor genes (PDK-2 by p53; PDK-4 by pRB). Furthermore, PDK-1 expression has been clinically correlated with poor prognosis in Gastric, HNSCC and Colon cancer. Previous studies employing RNAi have provided evidence for a survival role for PDK-1 as well as its importance in maintaining the glycolytic phenotype of cancer cells. DCA, a weak inhibitor of PDK, has also been used to study the role of PDK in cancer; however, interpretation of these studies has been complicated by conflicting data and the lack of specificity of this agent for PDK. Here we report the discovery of a novel, potent and selective pan-isoform inhibitor of PDK, VER-246608. Consistent with a PDK mediated MOA, treatment of PC-3 cells with VER-246608 resulted in increased PDC activity and oxygen consumption as well as reduced lactate production and glucose consumption. Interestingly, modulation of glycolytic activity required compound concentrations which achieved > 90 % reduction in E1α phosphorylation and this was only observed under glucose depleted conditions. Under normal culture conditions, VER-246608 showed little cytotoxicity to cancer cells. We hypothesised that the supraphysiological glucose concentrations present in cell culture media may limit the effect of PDK inhibition due to elevated intracellular pyruvate levels. We therefore performed cytotoxicity studies under conditions of limited nutrient availability. We found that combined depletion of glucose and glutamine or serum alone resulted in enhanced cytotoxicity vs normal media; however, hypoxic conditions had no effect. Furthermore, this differential cytotoxicity correlated with reduced pyruvate levels in the compound treated cells cultured in the above ‘austere’ conditions. VER-246608 also inhibited the growth of tumour spheroids with a potency that was comparable to cells grown in 2D culture. In addition, combination treatment studies revealed that VER-246608 potentiated anti-cancer agents which are known to influence mitochondrial function. In contrast, the lipoamide binding site inhibitor, Nov3r, showed no evidence of cytotoxicity under any of the above conditions and was ineffective in altering glycolytic activity. These studies suggest that PDK inhibition may be effective under the nutrient depleted conditions found in the tumour microenvironment and that combination treatments should be explored to reveal the full potential of this therapeutic strategy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B155. Citation Format: Jonathan Moore, Anna Staniszewska, Terence Shaw, Jalanie D'Alessandro, Ben Davis, Alan Surgenor, Lisa Baker, Natalia Massanova, James Murray, Alba Macias, Paul Brough, Mike Wood, Patrick C. Mahon. VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and demonstrates context-dependent cytotoxicity to cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B155.

Obtaining NMR assignments for slowly tumbling molecules such as detergent-solubilized membrane proteins is often compromised by low sensitivity as well as spectral overlap. Both problems can be addressed by amino-acid specific isotope labeling in conjunction with 15N–1H correlation experiments. In this work an extended combinatorial selective in vitro labeling scheme is proposed that seeks to reduce the number of samples required for assignment. Including three different species of amino acids in each sample, 15N, 1-13C, and fully 13C/15N labeled, permits identification of more amino acid types and sequential pairs than would be possible with previously published combinatorial methods. The new protocol involves recording of up to five 2D triple-resonance experiments to distinguish the various isotopomeric dipeptide species. The pattern of backbone NH cross peaks in this series of spectra adds a new dimension to the combinatorial grid, which otherwise mostly relies on comparison of [15N, 1H]–HSQC and possibly 2D HN(CO) spectra of samples with different labeled amino acid compositions. Application to two α-helical membrane proteins shows that using no more than three samples information can be accumulated such that backbone assignments can be completed solely based on 3D HNCA/HN(CO)CA experiments. Alternatively, in the case of severe signal overlap in certain regions of the standard suite of triple-resonance spectra acquired on uniformly labeled protein, or missing signals due to a lack of efficiency of 3D experiments, the remaining gaps can be filled.

For decades, molecular starting points for drug discovery have been found by screening large numbers of natural and synthetic compounds for biological activity in phenotypic and biochemical assays. Then, beginning in the mid to late 1990s, several pharmaceutical groups developed new approaches [such as structure/activity relationship by nuclear magnetic resonance (SAR by NMR), the SHAPES strategy, and needle screening] in which simple, low-molecular-weight compounds were screened for binding to the target of interest, and these relatively weak binding molecules were then used to systematically construct larger, more potent, drug leads. Such small screening molecules are now commonly called fragments and the related processes collectively called fragment-based lead discovery (FBLD).

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of two membrane proteins: calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a class B G-protein-coupled receptor (GPCR), possessing a characteristic large amino-terminal extracellular domain (ECD) important for ligand recognition and binding. Dimerization of CLR with RAMP1 provides specificity for CGRP versus related agonists. Here we report the expression, purification, and refolding of a soluble form of the CGRP receptor comprising a heterodimer of the CLR and RAMP1 ECDs. The extracellular protein domains corresponding to residues 23-133 of CLR and residues 26-117 of RAMP1 were shown to be sufficient for formation of a stable, monodisperse complex. The binding affinity of the purified ECD complex for the CGRP peptide was significantly lower than that of the native receptor (IC(50) of 12 microM for the purified ECD complex vs 233 pM for membrane-bound CGRP receptor), indicating that other regions of CLR and/or RAMP1 are important for peptide agonist binding. However, high-affinity binding to known potent and specific nonpeptide antagonists of the CGRP receptor, including olcegepant and telcagepant (K(D) < 0.02 muM), as well as N-terminally truncated peptides and peptide analogues (140 nM to 1.62 microM) was observed.

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.

Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell-cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl- ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate ( AMP) substrate. No liberation of [P-33] phosphate is observed from the gamma-P-33-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.

The discovery of new small-molecule chemical entities capable of modulating protein function usually begins with the high-throughput screening (HTS) of collections of compounds, or fragments of these, in order to select for molecules interacting with a target biomolecule (usually a protein). The process is the same whether the protein is well folded or is an intrinsically disordered one (IDP); however, subtle details may arise depending on the capability of the experimental technique to grasp particular structural and functional features of IDPs. For a full characterization of such interactions, the thermodynamic, stoichiometric, kinetic, and, quite often, structural details must be described. The set of biophysical methods now at hand constitutes a powerful toolbox for designing and implementing HTS assays and performing a full characterization of the interaction of hit and lead compounds selected by HTS. The aim of this chapter is to provide an overview of some high-throughput techniques used during the screening, identification, and validation of ligands. Fluorescence-based methods, which offer an attractive combination of high-throughput and small-cost but low-resolution structural aspects, and nuclear magnetic resonance (NMR)–based methods, which offer a combination of high-medium throughput and comprehensive structural insight, are discussed in detail, with recent examples applied to IDPs. We also describe briefly the use of surface plasmon resonance (SPR) and its complementarities with the techniques previously mentioned.

AbstractFor Abstract see ChemInform Abstract in Full Text.

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.