The discovery of quinoline-3-carboxamides as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors.

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50 = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50 = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50 = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.

Crystal structure of human CDK4 in complex with a D-type cyclin.

The cyclin D1-cyclin-dependent kinase 4 (CDK4) complex is a key regulator of the transition through the G(1) phase of the cell cycle. Among the cyclin/CDKs, CDK4 and cyclin D1 are the most frequently activated by somatic genetic alterations in multiple tumor types. Thus, aberrant regulation of the CDK4/cyclin D1 pathway plays an essential role in oncogenesis; hence, CDK4 is a genetically validated therapeutic target. Although X-ray crystallographic structures have been determined for various CDK/cyclin complexes, CDK4/cyclin D1 has remained highly refractory to structure determination. Here, we report the crystal structure of CDK4 in complex with cyclin D1 at a resolution of 2.3 A. Although CDK4 is bound to cyclin D1 and has a phosphorylated T-loop, CDK4 is in an inactive conformation and the conformation of the heterodimer diverges from the previously known CDK/cyclin binary complexes, which suggests a unique mechanism for the process of CDK4 regulation and activation.

X-ray Screening of an Electrophilic Fragment Library and Application toward the Development of a Novel ERK 1/2 Covalent Inhibitor.

Fragment-based drug discovery (FBDD) has become an established method for the identification of efficient starting points for drug discovery programs. In recent years, electrophilic fragment screening has garnered increased attention from both academia and industry to identify novel covalent hits for tool compound or drug development against challenging drug targets. Herein, we describe the design and characterization of an acrylamide-focused electrophilic fragment library and screening campaign against extracellular signal-regulated kinase 2 (ERK2) using high-throughput protein crystallography as the primary hit-finding technology. Several fragments were found to have covalently modified the adenosine triphosphate (ATP) binding pocket Cys166 residue. From these hits, 22, a covalent ATP-competitive inhibitor with improved potency (ERK2 IC50 = 7.8 µM), was developed.

Crystallographic screening using ultra-low-molecular-weight ligands to guide drug design.

We present a novel crystallographic screening methodology (MiniFrags) that employs high-concentration aqueous soaks with a chemically diverse and ultra-low-molecular-weight library (heavy atom count 5-7) to identify ligand-binding hot and warm spots on proteins. We propose that MiniFrag screening represents a highly effective method for guiding optimisation of fragment-derived lead compounds or chemical tools and that the high screening hit rates reflect enhanced sampling of chemical space.

Fragment-based screening using X-ray crystallography and NMR spectroscopy.

Approaches which start from a study of the interaction of very simple molecules (fragments) with the protein target are proving to be valuable additions to drug design. Fragment-based screening allows the complementarity between a protein active site and drug-like molecules to be rapidly and effectively explored, using structural methods. Recent improvements in the intensities of laboratory X-ray sources permits the collection of greater amounts of high-quality diffraction data and have been matched by developments in automation, crystallisation and data analysis. Developments in NMR screening, including the use of cryogenically cooled NMR probes and (19)F-containing reporter molecules have expanded the scope of this technique, while increasing the availability of binding site and quantitative affinity data for the fragments. Application of these methods has led to a greater knowledge of the chemical variety, structural features and energetics of protein-fragment interactions. While fragment-based screening has already been shown to reduce the timescales of the drug discovery process, a more detailed characterisation of fragment screening hits can reveal unexpected similarities between fragment chemotypes and protein active sites leading to improved understanding of the pharmacophores and the re-use of this information against other protein targets.

Biphenyl amide p38 kinase inhibitors 1: Discovery and binding mode.

The biphenyl amides (BPAs) are a novel series of p38 MAP kinase inhibitors. The discovery of the series through structure-based focused screening is described, and the binding mode of the compounds is explained with reference to X-ray crystal structures.

Application of fragment screening by X-ray crystallography to beta-secretase.

This paper describes an application of fragment screening to the aspartyl protease enzyme, beta-secretase (BACE-1), using high throughput X-ray crystallography. Three distinct chemotypes were identified by X-ray crystallography as binding to the catalytic aspartates either via an aminoheterocycle (such as 2-aminoquinoline), a piperidine, or an aliphatic hydroxyl group. The fragment hits were weak inhibitors of BACE-1 in the millimolar range but were of interest because most of them displayed relatively good ligand efficiencies. The aminoheterocycles exhibited a novel recognition motif that has not been seen before with aspartic proteases. Virtual screening around this motif identified an aminopyridine with increased potency and attractive growth points for further elaboration using structure-based drug design. The companion paper illustrates how sub-micromolar inhibitors were developed starting from this fragment.

Structures for the potential drug target purine nucleoside phosphorylase from Schistosoma mansoni causal agent of schistosomiasis.

Despite the availability of effective chemotherapy, schistosomiasis continues to be one of the major parasitic infections to affect the human population worldwide. Currently, little is known of the structural biology of the parasites that are responsible for the disease and few attempts have been made to develop second generation drugs, which may become essential if resistance to those currently available becomes an issue. Here, we describe three crystal structures for the enzyme purine nucleoside phosphorylase (PNP) from Schistosoma mansoni, a component of the purine salvage pathway. PNP is known to be essential for the recovery of purine bases and nucleosides in schistosomes, due to an absence of the enzymes for de novo synthesis, making it a sensitive point in the parasite's metabolism. In all three structures reported here, acetate occupies part of the base-binding site and is directly bound to the conserved glutamic acid at position 203. One of the structures presents the crystallization additive sulfobetaine 195 (NDSB195) occupying simultaneously the ribose and phosphate binding sites, whilst a second presents only phosphate in the latter. The observation of sulfobetaine specifically bound to the protein active site was unexpected and is unique to this structure as far as we are aware. Considerable flexibility is observed in the active site, principally due to variable structural disorder in the regions centered on residues 64 and 260. This conformational plasticity extends to the way in which both NDSB195 and phosphate bind to the individual monomers of the trimeric structure reported here. Differences between the parasite and human enzymes are limited principally to the base-binding site, where the substitution of V245 in the mammalian enzymes by S247 introduces additional hydrogen bonding potential to the site. This is satisfied in the structures described here by a water molecule whose presence is normally observed only in complexes with 6-oxopurines. Residue Y202, which replaces F200 in human PNP, is able to reach over the ribose-binding site to interact with H259 and is predicted to form an additional hydrogen bond with the 5' hydroxyl of nucleoside substrates.

Apo and inhibitor complex structures of BACE (beta-secretase).

Human BACE, also known as beta-secretase, shows promise as a potential therapeutic target for Alzheimer's disease. We determined the apo structure of BACE to 1.75 A, and a structure of a hydroxyethylamine inhibitor complex derived by soaking. These show significant active-site movements compared to previously described BACE structures. Additionally, the structures reveal two pockets that could be targeted by structure-based drug design.

Fragment-based lead discovery using X-ray crystallography.

Fragment screening offers an alternative to traditional screening for discovering new leads in drug discovery programs. This paper describes a fragment screening methodology based on high throughput X-ray crystallography. The method is illustrated against five proteins (p38 MAP kinase, CDK2, thrombin, ribonuclease A, and PTP1B). The fragments identified have weak potency (>100 microM) but are efficient binders relative to their size and may therefore represent suitable starting points for evolution to good quality lead compounds. The examples illustrate that a range of molecular interactions (i.e., lipophilic, charge-charge, neutral hydrogen bonds) can drive fragment binding and also that fragments can induce protein movement. We believe that the method has great potential for the discovery of novel lead compounds against a range of targets, and the companion paper illustrates how lead compounds have been identified for p38 MAP kinase starting from fragments such as those described in this paper.

Identification of novel p38alpha MAP kinase inhibitors using fragment-based lead generation.

We describe the structure-guided optimization of the molecular fragments 2-amino-3-benzyloxypyridine 1 (IC(50) 1.3 mM) and 3-(2-(4-pyridyl)ethyl)indole 2 (IC(50) 35 microM) identified using X-ray crystallographic screening of p38alpha MAP kinase. Using two separate case studies, the article focuses on the key compounds synthesized, the structure-activity relationships and the binding mode observations made during this optimization process, resulting in two potent lead series that demonstrate significant increases in activity. We describe the process of compound elaboration either through the growing out from fragments into adjacent pockets or through the conjoining of overlapping fragments and demonstrate that we have exploited the mobile conserved activation loop, consisting in part of Asp168-Phe169-Gly170 (DFG), to generate significant improvements in potency and kinase selectivity.

Fragment-Based Discovery of Potent and Selective DDR1/2 Inhibitors.

The DDR1 and DDR2 receptor tyrosine kinases are activated by extracellular collagen and have been implicated in a number of human diseases including cancer. We performed a fragment-based screen against DDR1 and identified fragments that bound either at the hinge or in the back pocket associated with the DFG-out conformation of the kinase. Modeling based on crystal structures of potent kinase inhibitors facilitated the "back-to-front" design of potent DDR1/2 inhibitors that incorporated one of the DFG-out fragments. Further optimization led to low nanomolar, orally bioavailable inhibitors that were selective for DDR1 and DDR2. The inhibitors were shown to potently inhibit DDR2 activity in cells but in contrast to unselective inhibitors such as dasatinib, they did not inhibit proliferation of mutant DDR2 lung SCC cell lines.

Crystal structure of human soluble adenylate cyclase reveals a distinct, highly flexible allosteric bicarbonate binding pocket.

Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,ß-methylene adenosine 5'-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein.

Structure of the BTB domain of Keap1 and its interaction with the triterpenoid antagonist CDDO.

The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.

Potent, selective inhibitors of fibroblast growth factor receptor define fibroblast growth factor dependence in preclinical cancer models.

We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role.

Crystal structure of Schistosoma purine nucleoside phosphorylase complexed with a novel monocyclic inhibitor.

A novel inhibitor of Schistosoma PNP was identified using an "in silico" approach allied to enzyme inhibition assays. The compound has a monocyclic structure which has not been previously described for PNP inhibitors. The crystallographic structure of the complex was determined and used to elucidate the binding mode within the active site. Furthermore, the predicted pose was very similar to that determined crystallographically, validating the methodology. The compound Sm_VS1, despite its low molecular weight, possesses an IC(50) of 1.3 microM, surprisingly low when compared with purine analogues. This is presumably due to the formation of eight hydrogen bonds with key residues in the active site E203, N245 and T244. The results of this study highlight the importance of the use of multiple conformations for the target during virtual screening. Indeed the Sm_VS1 compound was only identified after flipping the N245 side chain. It is expected that the structure will be of use in the development of new highly active non-purine based compounds against the Schistosoma enzyme.

Structure of a CBS-domain pair from the regulatory gamma1 subunit of human AMPK in complex with AMP and ZMP.

AMP-activated kinase (AMPK) is central to sensing energy status in eukaryotic cells via binding of AMP and ATP to CBS (cystathionine beta-synthase) domains in the regulatory gamma subunit. The structure of a CBS-domain pair from human AMPK gamma1 in complex with the physiological activator AMP and the pharmacological activator ZMP (AICAR) is presented.

The discovery of novel protein kinase inhibitors by using fragment-based high-throughput x-ray crystallography.

This article describes the application of a high-throughput X-ray crystallographic fragment-based screening methodology to identify low-molecular-weight leads for structure-based optimisation into protein kinase inhibitors. The identification of two novel p38alpha MAP kinase inhibitors (with IC50=65 and 150 nM) starting from low-molecular-weight fragments is described.

Cloning, expression and preliminary crystallographic studies of the potential drug target purine nucleoside phosphorylase from Schistosoma mansoni.

The parasite Schistosoma mansoni, unlike its mammalian hosts, lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. The gene encoding one enzyme of this pathway, purine nucleoside phosphorylase from S. mansoni (SmPNP) was identified, fully sequenced and cloned into the bacterial expression vector pMAL c2G to produce a protein in fusion with maltose-binding protein. The recombinant fusion protein was expressed at high levels and was purified in a single step by amylose resin affinity chromatography. After factor Xa cleavage, SmPNP was purified using a cation-exchange column and crystallized by hanging-drop vapour diffusion using polyethylene glycol 1500 as precipitant in the presence of 20% glycerol in acetate buffer. The use of the non-detergent sulfobetaine 195 (NDSB 195) as an additive had a marked effect on the size of the resulting crystals. Two data sets were obtained, one from a crystal grown in the absence of NDSB 195 and one from a crystal grown in its presence. The crystals are isomorphous and belong to the space group P2(1)2(1)2(1). It is intended to use the structures in the discovery and development of specific inhibitors of SmPNP.