Structure-Based Design of Selective LONP1 Inhibitors for Probing In Vitro Biology.

LONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure-based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology.

Identification of Potent and Selective RIPK2 Inhibitors for the Treatment of Inflammatory Diseases.

NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.

(R)-2-Phenylpyrrolidine Substituted Imidazopyridazines: A New Class of Potent and Selective Pan-TRK Inhibitors.

Deregulated kinase activities of tropomyosin receptor kinase (TRK) family members have been shown to be associated with tumorigenesis and poor prognosis in a variety of cancer types. In particular, several chromosomal rearrangements involving TRKA have been reported in colorectal, papillary thyroid, glioblastoma, melanoma, and lung tissue that are believed to be the key oncogenic driver in these tumors. By screening the Novartis compound collection, a novel imidazopyridazine TRK inhibitor was identified that served as a launching point for drug optimization. Structure guided drug design led to the identification of (R)-2-phenylpyrrolidine substituted imidazopyridazines as a series of potent, selective, orally bioavailable pan-TRK inhibitors achieving tumor regression in rats bearing KM12 xenografts. From this work the (R)-2-phenylpyrrolidine has emerged as an ideal moiety to incorporate in bicyclic TRK inhibitors by virtue of its shape complementarity to the hydrophobic pocket of TRKs.

Endovascular Treatment of Proximal Aortic Arch Lesions through a Retrograde Approach.

Tandem atherosclerotic lesions of the carotid bifurcation and the ipsilateral proximal common carotid artery (CCA) or innominate arteries (IA) can be challenging to treat. A surgical approach may treat the lesion at the carotid bifurcation, but proximal CCA or IA lesions require a major surgical exposure. An endovascular approach is challenging as well since anatomic variations, such as a type III aortic arch, can render navigation very difficult. We report our experience in the hybrid surgical and endovascular treatment of complex proximal CCA and IA lesions. Eleven patients who underwent hybrid procedures with surgical exposure (with or without endarterectomy) of the carotid artery and retrograde endovascular intervention of a proximal lesion were included in the study. The mean percentage of stenosis was 81%. Seven patients underwent a carotid endarterectomy (CEA), and 4 patients underwent only a surgical cutdown for retrograde endovascular access of the IA or left CCA. All procedures were technically successful. Eight patients had no symptoms within 30 days of the procedure. The hybrid retrograde endovascular approach through carotid exposure with or without CEA appears to be effective and safe in selected patients who have a high-risk complex anatomy of tandem lesions.

Radioprotective lightweight caps in the interventional cardiology setting: a randomised controlled trial (PROTECT).

AIMS: The aim of this study was to test the radioprotection efficacy and comfort of newer bilayer barium sulphate-bismuth oxide composite (XPF) caps in an interventional cardiology setting. METHODS AND RESULTS: Operators were randomly assigned to wear standard fabric (n=59), 0.3 mm (n=74), or 0.5 mm (n=64) lead-equivalent XPF caps. Radiation doses were measured by using dosimeters placed outside and underneath the caps. Wearing comfort was assessed at the end of each measurement on a visual analogue scale (VAS) (0-100, with 100 indicating optimal comfort). Procedural data did not differ between the XPF and standard groups. Mean standard, XPF 0.3 mm, and XPF 0.5 mm cap weights were 12.5 g, 118.4 g, and 123.7 g, respectively. VAS comfort ratings of the standard and XPF caps did not differ significantly (p=0.272). The mean radiation protection was 12.0%, 95% CI: 4.9-19.1% (standard caps, n=35), 91.5%, 95% CI: 87.4-95.6% (XPF 0.3 mm caps, n=45) and 97.1%, 95% CI: 92.5-100% (XPF 0.5 mm caps, n=44) (p≤0.001 for all group comparisons). Using the XPF caps, a cumulative total radiation dose reduction by almost factor 10 was evident (272 procedures, 22,310 µSv outside the XPF caps, 2,770 µSv inside the caps). CONCLUSIONS: Lightweight XPF caps show comparable comfort to standard fabric caps, but provide substantial radiation protection during fluoroscopy-guided cardiac interventions.

Endovascular treatment for traumatic scalp arteriovenous fistulas: results with Onyx embolization.

BACKGROUND: Arteriovenous fistulas of the scalp (S-AVFs) are rare lesions and may occur spontaneously or secondary to trauma. The use of Onyx for the treatment of S-AVFs is not well established at this time. We discuss three cases of traumatic S-AVFs treated successfully with Onyx embolization alone or in association with coils. METHODS: The database of patients treated at the Baptist Cardiac and Vascular Institute, Miami, Florida, was reviewed. All patients with traumatic S-AVFs treated with Onyx were included. RESULTS: Two men and one woman with progressive enlarging pulsatile mass with bruit or tinnitus had angiographic evidence of S-AVF and were treated. In two patients the S-AVFs were secondary to hair transplantation. They were treated with Onyx-18 embolization as the single treatment modality. One patient with S-AVF resulting from temporomandibular joint arthroscopy was treated with coils and subsequent Onyx-34 embolization. In one patient, transarterial microcatheterization and injection of Onyx-18 was performed. In another patient, the intra-arterial approach was prevented by arterial vessel tortuosity. Therefore, access to the fistula was obtained through direct puncture of a large frontal vein; contrast injection confirmed the positioning of the needle within the draining vein of the AVF and Onyx-18 was then injected while the outflow vein was compressed. In the third patient in this series, coils were deployed to allow safer and more controlled injection of Onyx-34. No procedure related complications were noted. Post-embolization angiography demonstrated successful and complete occlusion of the AVF immediately after treatment. Follow-up revealed complete resolution of the symptoms. CONCLUSIONS: Our experience in this small series indicates that endovascular treatment of S-AVFs with Onyx is rapid, safe, and highly effective.

Alberta Stroke Program Early CT Scale evaluation of multimodal computed tomography in predicting clinical outcomes of stroke patients treated with aspiration thrombectomy.

BACKGROUND AND PURPOSE: Patient selection is crucial in the endovascular treatment of acute ischemic stroke patients. Baseline computed tomographic (CT) images, evaluated with the Alberta Stroke Program Early CT Scale (ASPECTS), are considered significant predictors of outcome. In this study, we evaluated CT images and perfusion parameters, analyzed with ASPECTS, as final outcome predictors after endovascular stroke treatment. METHODS: We analyzed a cohort of patients with acute ischemic stroke and endovascular treatment. Patients with an occlusion of the M1 segment and multimodal CT imaging were included. CT perfusion data were reconstructed using commercial software. Two experienced neuroradiologists separately reviewed and scored CT and CT perfusion images with the ASPECTS score. Parameters were compared between patients with poor and with favorable follow-up outcome. Significantly different variables were further analyzed by forward stepwise logistic regression. RESULTS: Fifty-one patients were included in our study. Baseline characteristics did not differ between patients with favorable and poor outcomes. No significant difference in recanalization status, the various times, or CT ASPECTS was demonstrated between these 2 groups. Significant differences were demonstrated for age (P=0.0049), cerebral blood volume ASPECTS (P=0.0007), and between cerebral blood volume and cerebral blood flow ASPECTS (P=0.0045). Cerebral blood volume ASPECTS>7 demonstrated the highest sensitivity and specificity for favorable outcome with 84% and 79%, respectively. CONCLUSIONS: CT perfusion parameters, evaluated with ASPECTS, are optimal predictors of outcome and are more sensitive and specific than CT ASPECTS in the prediction of favorable outcome. Use of these parameters in treatment decisions could reduce futile recanalizations.

Techniques and results--penumbra aspiration catheter.

The Penumbra System is an endovascular mechanical thrombectomy device that uses continuous aspiration to perform recanalization of occluded intracranial vessels. The objective of this article was to provide a comprehensive overview of the data on technical and functional outcome so far published for aspiration thrombectomy and resume techniques that can be used to optimize functionality of the Penumbra System. We focus on existing clinical data as well as our institutional experience and techniques.

Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima.

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-ß-D-xylopyranoside monoacetates as substrates in a ß-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic α/ß-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.

Discovery of GNF-5837, a Selective TRK Inhibitor with Efficacy in Rodent Cancer Tumor Models.

Neurotrophins and their receptors (TRKs) play key roles in the development of the nervous system and the maintenance of the neural network. Accumulating evidence points to their role in malignant transformations, chemotaxis, metastasis, and survival signaling and may contribute to the pathogenesis of a variety of tumors of both neural and non-neural origin. By screening the GNF kinase collection, a series of novel oxindole inhibitors of TRKs were identified. Optimization led to the identification of GNF-5837 (22), a potent, selective, and orally bioavailable pan-TRK inhibitor that inhibited tumor growth in a mouse xenograft model derived from RIE cells expressing both TRKA and NGF. The properties of 22 make it a good tool for the elucidation of TRK biology in cancer and other nononcology indications.

Structure and ligand binding of the soluble domain of a Thermotoga maritima membrane protein of unknown function TM1634.

As a part of the Joint Center for Structural Genomics (JCSG) biological targets, the structures of soluble domains of membrane proteins from Thermotoga maritima were pursued. Here, we report the crystal structure of the soluble domain of TM1634, a putative membrane protein of 128 residues (15.1 kDa) and unknown function. The soluble domain of TM1634 is an alpha-helical dimer that contains a single tetratrico peptide repeat (TPR) motif in each monomer where each motif is similar to that found in Tom20. The overall fold, however, is unique and a DALI search does not identify similar folds beyond the 38-residue TPR motif. Two different putative ligand binding sites, in which PEG200 and Co(2+) were located, were identified using crystallography and NMR, respectively.

Crystal structure of human Pus10, a novel pseudouridine synthase.

Pseudouridine (Psi) synthases catalyze the formation of one or more specific Psis in structured RNAs. Five families of Psi synthases have been characterized based on sequence homology. Pus10 has no significant sequence homology to these defined families and therefore represents a new family of Psi synthases. Initial characterization studies show that an archael Pus10 catalyzes the universally conserved Psi55 in tRNA. We present here the crystal structure of human Pus10 at 2.0 A resolution, which is the first structural description from this novel Psi synthase family. Pus10 is a crescent-shaped molecule with two domains, the universally conserved Psi synthase catalytic domain and a THUMP-containing domain, which is unique to the Pus10 family. Superposition of the catalytic domains of Pus10 and other Psi synthases identifies the full set of conserved Psi synthase active site residues indicating that Pus10 likely employs a similar catalytic mechanism to other Psi synthases. The Pus10 active site is located in a deep pocket of a basic cleft adjacent to flexible thumb and forefinger loops, which could provide further stabilization for binding the RNA substrate. Modeling studies demonstrate that the cleft between the catalytic and accessory domain is large enough and electrostatically compatible to accommodate an RNA stem and support the role of the N-terminal domain as an accessory RNA-binding domain.

The nuclear receptor LXR is a glucose sensor.

The liver has a central role in glucose homeostasis, as it has the distinctive ability to produce and consume glucose. On feeding, glucose influx triggers gene expression changes in hepatocytes to suppress endogenous glucose production and convert excess glucose into glycogen or fatty acids to be stored in adipose tissue. This process is controlled by insulin, although debate exists as to whether insulin acts directly or indirectly on the liver. In addition to stimulating pancreatic insulin release, glucose also regulates the activity of ChREBP, a transcription factor that modulates lipogenesis. Here we describe another mechanism whereby glucose determines its own fate: we show that glucose binds and stimulates the transcriptional activity of the liver X receptor (LXR), a nuclear receptor that coordinates hepatic lipid metabolism. d-Glucose and d-glucose-6-phosphate are direct agonists of both LXR-alpha and LXR-beta. Glucose activates LXR at physiological concentrations expected in the liver and induces expression of LXR target genes with efficacy similar to that of oxysterols, the known LXR ligands. Cholesterol homeostasis genes that require LXR for expression are upregulated in liver and intestine of fasted mice re-fed with a glucose diet, indicating that glucose is an endogenous LXR ligand. Our results identify LXR as a transcriptional switch that integrates hepatic glucose metabolism and fatty acid synthesis.

Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution.

Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.

Crystal structure of the human TRPV2 channel ankyrin repeat domain.

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 A resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.