Target 2035 - an update on private sector contributions.

Target 2035, an international federation of biomedical scientists from the public and private sectors, is leveraging 'open' principles to develop a pharmacological tool for every human protein. These tools are important reagents for scientists studying human health and disease and will facilitate the development of new medicines. It is therefore not surprising that pharmaceutical companies are joining Target 2035, contributing both knowledge and reagents to study novel proteins. Here, we present a brief progress update on Target 2035 and highlight some of industry's contributions.

Target 2035 - update on the quest for a probe for every protein.

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.

Biophysics in drug discovery: impact, challenges and opportunities.

Over the past 25 years, biophysical technologies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, surface plasmon resonance spectroscopy and isothermal titration calorimetry have become key components of drug discovery platforms in many pharmaceutical companies and academic laboratories. There have been great improvements in the speed, sensitivity and range of possible measurements, providing high-resolution mechanistic, kinetic, thermodynamic and structural information on compound-target interactions. This Review provides a framework to understand this evolution by describing the key biophysical methods, the information they can provide and the ways in which they can be applied at different stages of the drug discovery process. We also discuss the challenges for current technologies and future opportunities to use biophysical methods to solve drug discovery problems.

Structural analysis of human KDM5B guides histone demethylase inhibitor development.

Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe(2+)-dependent oxygenases that act as histone H3K4 demethylases, thereby regulating cell proliferation and stem cell self-renewal and differentiation. Here we report crystal structures of the catalytic core of the human KDM5B enzyme in complex with three inhibitor chemotypes. These scaffolds exploit several aspects of the KDM5 active site, and their selectivity profiles reflect their hybrid features with respect to the KDM4 and KDM6 families. Whereas GSK-J1, a previously identified KDM6 inhibitor, showed about sevenfold less inhibitory activity toward KDM5B than toward KDM6 proteins, KDM5-C49 displayed 25-100-fold selectivity between KDM5B and KDM6B. The cell-permeable derivative KDM5-C70 had an antiproliferative effect in myeloma cells, leading to genome-wide elevation of H3K4me3 levels. The selective inhibitor GSK467 exploited unique binding modes, but it lacked cellular potency in the myeloma system. Taken together, these structural leads deliver multiple starting points for further rational and selective inhibitor design.

Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor.

Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin.

As a key regulator of mitosis, the Ser/Thr protein polo-like kinase-1 (Plk-1) is a well validated drug target in cancer therapy. In order to enable structure-guided drug design, determination of the crystal structure of the kinase domain of Plk-1 was attempted. Using a multi-parallel cloning and expression approach, a set of length variants were identified which could be expressed in large amounts from insect cells and which could be purified to high purity. However, all attempts to crystallize these constructs failed. Crystals were ultimately obtained by generating designed ankyrin-repeat proteins (DARPins) selective for Plk-1 and using them for cocrystallization. Here, the first crystal structure of the kinase domain of wild-type apo Plk-1, in complex with DARPin 3H10, is presented, underlining the power of selective DARPins as crystallization tools. The structure was refined to 2.3 A resolution and shows the active conformation of Plk-1. It broadens the basis for modelling and cocrystallization studies for drug design. The binding epitope of 3H10 is rich in arginine, glutamine and lysine residues, suggesting that the DARPin enabled crystallization by masking a surface patch which is unfavourable for crystal contact formation. Based on the packing observed in the crystal, a truncated DARPin variant was designed which showed improved binding characteristics.

A structural biology view of target drugability.

BACKGROUND: With long and costly drug development times there is a need in the pharmaceutical industry to prioritize targets early in the drug discovery process. One of the possible criteria is 'protein drugability', a term with multiple understandings in the literature. Among others, it is the likelihood of finding a selective, low-molecular weight molecule that binds with high affinity to the target. OBJECTIVE: Which methods are available for drugability prediction? What can be achieved by such predictions and how can they influence the target prioritization process? METHODS: The main focus is on sequence- and structure-related computational methods for drugability prediction, giving an overview on their background as well as their bias and limitations with an emphasis on the structural biology point of view. RESULTS/CONCLUSION: Structural drugability assessment presents one criterion for prioritization of a target portfolio by enabling classification of targets into low, average, or high drugability.

Structural basis for a high affinity inhibitor bound to protein kinase MK2.

The Ser/Thr protein kinase MAPKAP kinase 2 (MK2) plays a crucial role in inflammation. We determined the structure of the kinase domain of MK2 in complex with a low molecular mass inhibitor in two different crystal forms, obtained from soaking and co-crystallization. To our knowledge, these are the first structures of MK2 showing the binding mode of an inhibitor with high binding affinity (IC50 8.5 nM). The two crystal forms revealed conformational flexibility in the binding site and extend the experimental basis for rational drug design. Crystal form-1 contained one MK2 molecule per asymmetric unit. Form-2 contained 12 molecules, which arrange into two different types of MK2 trimers. One of them may serve as a model for an intermediate state during substrate phosphorylation, as each MK2 monomer places its activation segment into the substrate peptide binding groove of the trimer neighbor.

Compartmentalization of a unique ADP/ATP carrier protein SFEC (Sperm Flagellar Energy Carrier, AAC4) with glycolytic enzymes in the fibrous sheath of the human sperm flagellar principal piece.

The longest part of the sperm flagellum, the principal piece, contains the fibrous sheath, a cytoskeletal element unique to spermiogenesis. We performed mass spectrometry proteomics on isolated human fibrous sheaths identifying a unique ADP/ATP carrier protein, SFEC [AAC4], seven glycolytic enzymes previously unreported in the human sperm fibrous sheath, and sorbitol dehydrogenase. SFEC, pyruvate kinase and aldolase were co-localized by immunofluorescence to the principal piece. A homology model constructed for SFEC predicted unique residues at the entrance to the nucleotide binding pocket of SFEC that are absent in other human ADP/ATP carriers, suggesting opportunities for selective drug targeting. This study provides the first evidence of a role for an ADP/ATP carrier family member in glycolysis. The co-localization of SFEC and glycolytic enzymes in the fibrous sheath supports a growing literature that the principal piece of the flagellum is capable of generating and regulating ATP independently from mitochondrial oxidation in the mid-piece. A model is proposed that the fibrous sheath represents a highly ordered complex, analogous to the electron transport chain, in which adjacent enzymes in the glycolytic pathway are assembled to permit efficient flux of energy substrates and products with SFEC serving to mediate energy generating and energy consuming processes in the distal flagellum, possibly as a nucleotide shuttle between flagellar glycolysis, protein phosphorylation and mechanisms of motility.

Identifying protein construct variants with increased crystallization propensity--a case study.

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.

The chemoresistance gene ABCG2 (MXR/BCRP1/ABCP1) is not expressed in melanomas but in single neuroendocrine carcinomas of the skin.

BACKGROUND: As the molecular mechanisms in melanoma chemoresistance remain unknown to date, we hypothesized these tumors to express the ATP-binding cassette (ABC) transporter G2 (ABCG2/MXR/BCRP1/ABCP1), a recently detected membrane transporter and putative stem-cell marker. Besides melanoma, we addressed the neuroendocrine carcinoma of the skin (Merkel cell carcinoma), another cutaneous cancer supposed to originate from neuroectoderm and usually chemoresistant. METHODS AND RESULTS: Upon semiquantitative reverse transcription polymerase chain reaction, ABCG2 mRNA expression was not upregulated in 18 melanoma resection specimens when compared with 19 acquired melanocytic nevi from which melanomas are known to often arise (Mantel-Haenszel test, p=0.3). At protein level, immunohistochemistry was negative in all 66 investigated melanoma resection specimens (50 primary melanomas and 16 cutaneous/subcutaneous metastases) and in 19 acquired melanocytic nevi. Among 29 neuroendocrine carcinomas of the skin, ABCG2 protein was detected in single clusters of cells in three tumors. As a positive control, three dermatofibrosarcomas were also stained and showed ABCG2 protein expression of the endothelial cells of the blood vessels. CONCLUSION: Altogether, chemoresistance of melanomas and neuroendocrine carcinomas of the skin cannot be explained by expression of the ABCG2-chemoresistance gene. Most of these tumors do not exhibit this potential stem-cell feature.

The target discovery process.

In order to minimise attrition rates in drug development projects, a target discovery process is implemented to select and characterise the most suitable candidate kinase targets, before lead identification and lead optimisation are embarked upon. The process consists of 1) target selection, 2) target assessment, and 3) target validation. This rational approach to target discovery, as a prerequisite for lead discovery, ensures that new therapeutic targets fulfil a set of general criteria, as well as indication-specific, descriptive and functional ones. The approach should ultimately maximise the likelihood of achieving target-selective inhibition by small-molecule inhibitors with minimal in vivo side effects and a therapeutic effect based on a sound biological hypothesis.

Adhesion molecules CD171 (L1CAM) and CD24 are expressed by primary neuroendocrine carcinomas of the skin (Merkel cell carcinomas).

BACKGROUND: The neuroendocrine carcinoma of the skin is a rare malignant neuroendocrine tumor, which frequently metastasizes in regional lymph nodes or visceral organs. As adhesive interactions with endothelia, leukocytes, or thrombocytes enable malignant cells to penetrate the endothelium and to circulate in blood or lymphatic vessels, we here addressed the adhesion molecules CD171 (L1CAM) and CD24, which are known to be expressed by neurons, neuroblastomas, and other malignant tumors. METHODS: Thirty-one neuroendocrine carcinomas of the skin (22 primary tumors, four recurrent tumors, and five metastases) were included in the study. Immunohistochemical staining of CD171 and CD24 was performed by the streptavidin-biotin-peroxidase-complex technique and a nickel-enhanced diaminobenzidine (DAB) reaction using the monoclonal antibodies UJ 127.11 and ML-5, respectively. RESULTS: CD171 expression was detected in most neuroendocrine carcinomas of the skin, and staining was less frequent in metastases and recurrences in comparison with primary tumors which was statistically significant. The majority of neuroendocrine carcinomas of the skin was also positive for the mucin-like adhesion protein CD24. In contrast to tumor cells, cytokeratin 20-positive Merkel cells in 12 trichoblastomas and one fibroepithelioma of Pinkus were all negative for CD171 and CD24 staining. CONCLUSIONS: Expression of CD171 and CD24 is found in most neuroendocrine carcinomas of the skin, which may be used diagnostically. Further studies will assess whether this feature may contribute to metastasis of neuroendocrine carcinomas of the skin by facilitating transendothelial migration or tumor cell dissemination as has been suggested for other malignancies.

Structural basis for the glucocorticoid response in a mutant human androgen receptor (AR(ccr)) derived from an androgen-independent prostate cancer.

The crystal structure of a mutant androgen receptor (AR) ligand-binding domain (LBD) in complex with the agonist 9alpha-fluorocortisol has been determined at 1.95 A resolution. This mutant AR contains two mutations (L701H and T877A) and was previously reported as a high-affinity cortisol/cortisone responsive AR (AR(ccr)) isolated from the androgen-independent human prostate cancer cell lines MDA PCa 2a and 2b (Zhao et al. Nature Med. 2000, 6, 703-6). The three-dimensional structure of the AR(ccr) LBD complexed with 9alpha-fluorocortisol shows the typical conformation of an agonist-bound nuclear receptor in which helix 12 is precisely positioned as a "lid" for the ligand-binding pocket. Binding of 9alpha-fluorocortisol to the AR(ccr) involves favorable hydrogen bond patterns on the C17 and C21 substituents of the ligand due to the mutations at 701 and 877 in the AR(ccr). Our studies provide the first structural explanation for the glucocorticoid activation of AR(ccr), which is important for the development of new therapeutic treatments for androgen-independent prostate cancer.