Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides.

Dampening functional levels of the mitochondrial deubiquitylating enzyme Ubiquitin-specific protease 30 (USP30) has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson's Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria, which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide-containing compound, USP30inh. Activity-based protein profiling mass spectrometry confirmed target engagement, high selectivity, and potency of USP30inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30inh enzyme kinetics inferred slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with USP30inh, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30inh binds to this thumb-palm cleft, which guides the ubiquitin C terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma.

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

Studying RNA-Protein Complexes Using X-Ray Crystallography.

A wide range of biological processes rely on complexes between ribonucleic acids (RNAs) and proteins. Determining the three-dimensional structures of RNA-protein complexes is crucial to elucidate the relationship between structure and biological function. X-ray crystallography represents the most widely used technique to characterize RNA-protein complexes at atomic resolution; however, determining their three-dimensional structures remains challenging. RNase contamination can ruin crystallization experiments by degrading RNA in complex with protein, leading to sample heterogeneity, and the conformational flexibility inherent in both protein and RNA can limit crystallizability. Furthermore, the three-dimensional structure can be difficult to accurately model at the typical diffraction limit of 2.5 Å resolution or lower for RNA-protein complex crystals. At this resolution, phosphates, which are electron dense, and bases, which are large, rigid, and planar, tend to be well resolved and easy to position in the electron density map, whereas other features, e.g., sugar atoms, can be difficult to accurately position. This chapter focuses on methods that can be used to overcome the unique problems faced when crystallizing RNA-protein complexes and determining their three-dimensional structures using X-ray crystallography.

A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors.

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

Molecular basis of USP7 inhibition by selective small-molecule inhibitors.

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

Structure-Based Design of Potent and Selective Inhibitors of the Metabolic Kinase PFKFB3.

A weak screening hit with suboptimal physicochemical properties was optimized against PFKFB3 kinase using critical structure-guided insights. The resulting compounds demonstrated high selectivity over related PFKFB isoforms and modulation of the target in a cellular context. A selected example demonstrated exposure in animals following oral dosing. Examples from this series may serve as useful probes to understand the emerging biology of this metabolic target.

Saturated gain spectrum of VECSELs determined by transient measurement of lasing onset.

We describe time-resolved measurements of the evolution of the spectrum of radiation emitted by an optically-pumped continuous-wave InGaAs-GaAs quantum well laser, recorded as lasing builds up from noise to steady state. We extract a fitting parameter corresponding to the gain dispersion of the parabolic spectrum equal to -79 ± 30 fs2 and -36 ± 6 fs2 for a resonant and anti-resonant structure, respectively. Furthermore the recorded evolution of the spectrum allows for the calculation of an effective FWHM gain bandwidth for each structure, of 11 nm and 18 nm, respectively.

Morpholylureas are a new class of potent and selective inhibitors of the type 5 17-ß-hydroxysteroid dehydrogenase (AKR1C3).

Inhibitors of the aldo-keto reductase enzyme AKR1C3 are of interest as potential drugs for leukemia and hormone-related cancers. A series of non-carboxylate morpholino(phenylpiperazin-1-yl)methanones were prepared by palladium-catalysed coupling of substituted phenyl or pyridyl bromides with the known morpholino(piperazin-1-yl)methanone, and shown to be potent (IC50∼100nM) and very isoform-selective inhibitors of AKR1C3. Lipophilic electron-withdrawing substituents on the phenyl ring were positive for activity, as was an H-bond acceptor on the other terminal ring, and the ketone moiety (as a urea) was essential. These structure-activity relationships are consistent with an X-ray structure of a representative compound bound in the AKR1C3 active site, which showed H-bonding between the carbonyl oxygen of the drug and Tyr55 and His117 in the 'oxyanion hole' of the enzyme, with the piperazine bridging unit providing the correct twist to allow the terminal benzene ring to occupy the lipophilic pocket and align with Phe311.

Studying protein-ligand interactions using X-ray crystallography.

X-ray crystallography is a powerful technique for studying protein-ligand interactions. Advances in techniques have meant that it is now possible to routinely determine the structures of ligand complexes in the majority of cases where crystallization conditions and protein structures are already known. Ligand soaking or cocrystallization, together with the potential use of molecular replacement, provides data for determining the structures of a protein in complex with ligands. Furthermore, advances in protein structure model building facilitate automatic ligand fitting to residual electron density in the protein-ligand complex.

Synthesis and structure-activity relationships for 1-(4-(piperidin-1-ylsulfonyl)phenyl)pyrrolidin-2-ones as novel non-carboxylate inhibitors of the aldo-keto reductase enzyme AKR1C3.

High expression of the aldo-keto reductase enzyme AKR1C3 in the human prostate and breast has implicated it in the development and progression of leukemias and of prostate and breast cancers. Inhibitors are thus of interest as potential drugs. Most inhibitors of AKR1C3 are carboxylic acids, whose transport into cells is likely dominated by carrier-mediated processes. We describe here a series of (piperidinosulfonamidophenyl)pyrrolidin-2-ones as potent (<100 nM) and isoform-selective non-carboxylate inhibitors of AKR1C3. Structure-activity relationships identified the sulfonamide was critical, and a crystal structure showed the 2-pyrrolidinone does not interact directly with residues in the oxyanion hole. Variations in the position, co-planarity or electronic nature of the pyrrolidinone ring severely diminished activity, as did altering the size or polarity of the piperidino ring. There was a broad correlation between the enzyme potencies of the compounds and their effectiveness at inhibiting AKR1C3 activity in cells.

Structural and functional conservation profiles of novel cathepsin L-like proteins identified in the Drosophila melanogaster genome.

Cathepsin L is a cysteine protease which degrades connective tissue proteins including collagen, elastin, and fibronectin. In this study, five well-characterized cathepsin L proteins from different arthropods were used as query sequences for the Drosophila genome database. The search yielded 10 cathepsin L-like sequences, of which eight putatively represent novel cathepsin L-like proteins. To understand the phylogenetic relationship among these cathepsin L-like proteins, a phylogenetic tree was constructed based on their sequences. In addition, models of the tertiary structures of cathepsin L were constructed using homology modeling methods and subjected to molecular dynamics simulations to obtain reasonable structure to understand its dynamical behavior. Our findings demonstrate that all of the potential Drosophila cathepsin L-like proteins contain at least one cathepsin propeptide inhibitor domain. Multiple sequence alignment and homology models clearly highlight the conservation of active site residues, disulfide bonds, and amino acid residues critical for inhibitor binding. Furthermore, comparative modeling indicates that the sequence/structure/function profiles and active site architectures are conserved.

Crystal structures of three classes of non-steroidal anti-inflammatory drugs in complex with aldo-keto reductase 1C3.

Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.

3-(3,4-Dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic Acids: highly potent and selective inhibitors of the type 5 17-ß-hydroxysteroid dehydrogenase AKR1C3.

A high-throughput screen identified 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acid as a novel, highly potent (low nM), and isoform-selective (1500-fold) inhibitor of aldo-keto reductase AKR1C3: a target of interest in both breast and prostate cancer. Crystal structure studies showed that the carboxylate group occupies the oxyanion hole in the enzyme, while the sulfonamide provides the correct twist to allow the dihydroisoquinoline to bind in an adjacent hydrophobic pocket. SAR studies around this lead showed that the positioning of the carboxylate was critical, although it could be substituted by acid isosteres and amides. Small substituents on the dihydroisoquinoline gave improvements in potency. A set of "reverse sulfonamides" showed a 12-fold preference for the R stereoisomer. The compounds showed good cellular potency, as measured by inhibition of AKR1C3 metabolism of a known dinitrobenzamide substrate, with a broad rank order between enzymic and cellular activity, but amide analogues were more effective than predicted by the cellular assay.

Targeting cancer using fragment based drug discovery.

Over the past decade, fragment-based drug discovery has developed significantly and has gained increasing popularity in the pharmaceutical industry as a powerful alternative and complement to traditional high-throughput screening approaches for hit identification. Fragment-based methods are capable of rapidly identifying starting points for structure-based drug design from relatively small libraries of low molecular weight compounds. The main constraints are the need for sensitive methods that can reliably detect the typically weak interactions between fragments and the target protein, and strategies for transforming fragments into higher molecular weight drug candidates. This approach has recently been validated as series of compounds from various programs have entered clinical trials.

Structural and functional characterization of human Iba proteins.

Iba2 is a homolog of ionized calcium-binding adapter molecule 1 (Iba1), a 17-kDa protein that binds and cross-links filamentous actin (F-actin) and localizes to membrane ruffles and phagocytic cups. Here, we present the crystal structure of human Iba2 and its homodimerization properties, F-actin cross-linking activity, cellular localization and recruitment upon bacterial invasion in comparison with Iba1. The Iba2 structure comprises two central EF-hand motifs lacking bound Ca2+. Iba2 crystallized as a homodimer stabilized by a disulfide bridge and zinc ions. Analytical ultracentrifugation revealed a different mode of dimerization under reducing conditions that was independent of Ca2+. Furthermore, no binding of Ca2+ up to 0.1 mM was detected by equilibrium dialysis. Correspondingly, Iba EF-hand motifs lack residues essential for strong Ca2+ coordination. Sedimentation experiments and microscopy detected pronounced, indistinguishable F-actin binding and cross-linking activity of Iba1 and Iba2 with induction of F-actin bundles. Fluorescent Iba fusion proteins were expressed in HeLa cells and co-localized with F-actin. Iba1 was recruited into cellular projections to a larger extent than Iba2. Additionally, we studied Iba recruitment in a Shigella invasion model that induces cytoskeletal rearrangements. Both proteins were recruited into the bacterial invasion zone and Iba1 was again concentrated slightly higher in the cellular extensions.

Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily.

The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.1 A resolution. The enzyme forms an oval-shaped ring of four subunits, each consisting of an N-terminal domain with a four-stranded beta-sheet flanked by two alpha-helices, and a C-terminal catalytic domain with a fumarylacetoacetate hydrolase (FAH) fold. Crystal structures of complexes of the enzyme with magnesium or calcium ions and either a substrate analog 2-oxobutyrate, or the aldehyde enzyme product 2,5-dioxopentanoate revealed that the divalent metal ion in the active site is coordinated octahedrally by three conserved carboxylate residues, a water molecule, and both the carboxylate and the oxo groups of the substrate molecule. An enzymatic mechanism for base-catalyzed dehydration is proposed on the basis of the binding mode of the substrate to the metal ion, which suggests that the enzyme enhances the acidity of the protons alpha to the carbonyl group, facilitating their abstraction by glutamate 114. A comprehensive structural comparison of members of the FAH superfamily is presented and their evolution is discussed, providing a basis for functional investigations of this largely unexplored protein superfamily.

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.

Automated technologies and novel techniques to accelerate protein crystallography for structural genomics.

The sequence infrastructure that has arisen through large-scale genomic projects dedicated to protein analysis, has provided a wealth of information and brought together scientists and institutions from all over the world. As a consequence, the development of novel technologies and methodologies in proteomics research is helping to unravel the biochemical and physiological mechanisms of complex multivariate diseases at both a functional and molecular level. In the late sixties, when X-ray crystallography had just been established, the idea of determining protein structure on an almost universal basis was akin to an impossible dream or a miracle. Yet only forty years after, automated protein structure determination platforms have been established. The widespread use of robotics in protein crystallography has had a huge impact at every stage of the pipeline from protein cloning, over-expression, purification, crystallization, data collection, structure solution, refinement, validation and data management- all of which have become more or less automated with minimal human intervention necessary. Here, recent advances in protein crystal structure analysis in the context of structural genomics will be discussed. In addition, this review aims to give an overview of recent developments in high throughput instrumentation, and technologies and strategies to accelerate protein structure/function analysis.