Fragment-Guided Discovery of Pyrazole Carboxylic Acid Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2 Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction.

The NRF2-mediated cytoprotective response is central to cellular homoeostasis, and there is increasing interest in developing small-molecule activators of this pathway as therapeutics for diseases involving chronic oxidative stress. The protein KEAP1, which regulates NRF2, is a key point for pharmacological intervention, and we recently described the use of fragment-based drug discovery to develop a tool compound that directly disrupts the protein-protein interaction between NRF2 and KEAP1. We now present the identification of a second, chemically distinct series of KEAP1 inhibitors, which provided an alternative chemotype for lead optimization. Pharmacophoric information from our original fragment screen was used to identify new hit matter through database searching and to evolve this into a new lead with high target affinity and cell-based activity. We highlight how knowledge obtained from fragment-based approaches can be used to focus additional screening campaigns in order to de-risk projects through the rapid identification of novel chemical series.

Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

Accelerated real-time cardiac MRI using iterative sparse SENSE reconstruction: comparing performance in patients with sinus rhythm and atrial fibrillation.

OBJECTIVES: To compare accelerated real-time cardiac MRI (CMR) using sparse spatial and temporal undersampling and non-linear iterative SENSE reconstruction (RT IS SENSE) with real-time CMR (RT) and segmented CMR (SEG) in a cohort that included atrial fibrillation (AF) patients. METHODS: We evaluated 27 subjects, including 11 AF patients, by acquiring steady-state free precession cine images covering the left ventricle (LV) at 1.5 T with SEG (acceleration factor 2, TR 42 ms, 1.8 × 1.8 × 6 mm3), RT (acceleration factor 3, TR 62 ms, 3.0 × 3.0 × 7 mm3), and RT IS SENSE (acceleration factor 9.9-12, TR 42 ms, 2.0 × 2.0 × 7 mm3). We performed quantitative LV functional analysis in sinus rhythm (SR) patients and qualitatively scored image quality, noise and artefact using a 5-point Likert scale in the complete cohort and AF and SR subgroups. RESULTS: There was no difference between LV functional parameters between acquisitions in SR patients. RT IS SENSE short-axis image quality was superior to SEG (4.5 ± 0.6 vs. 3.9 ± 1.1, p = 0.007) and RT (3.8 ± 0.4, p = 0.003). There was reduced artefact in RT IS SENSE compared to SEG (4.4 ± 0.6 vs. 3.8 ± 1.2, p = 0.04), driven by arrhythmia performance. RT IS SENSE short-axis image quality was superior to SEG (4.6 ± 0.5 vs. 3.1 ± 1.0, p < 0.001) in the AF subgroup. CONCLUSION: Accelerated real-time CMR with iterative sparse SENSE provides excellent clinical performance, especially in patients with AF. KEY POINTS: • Iterative sparse SENSE significantly accelerates real-time cardiovascular MRI acquisitions. • It provides excellent qualitative and quantitative performance in sinus rhythm patients. • It outperforms standard segmented acquisitions in patients with atrial fibrillation. • It improves the trade-off between temporal and spatial resolution in real-time imaging.

The consistency of myocardial strain derived from heart deformation analysis.

The purpose of this study was to assess the consistency of semi-automated myocardial strain analysis by prototype software across field strengths, temporal resolutions, and examinations. 35 volunteers (48 ± 13 years; 20% women) and 25 patients (54 ± 12 years; 44% women) without significant cardiac dysfunction underwent cine cardiac magnetic resonance imaging (CMR) at 1.5 T with a temporal resolution of 39.2 msec. 34 subjects also underwent imaging at 3.0 T; 16 had repeat examinations within 14 days; and 9 underwent CMR with temporal resolutions of 12.5 and 39.2 msec on the same day. Prototype heart deformation analysis (HDA) software was used to retrospectively quantify strain from segmented balanced steady state free precession (bSSFP) cinegraphic images. Myocardial contours were automatically generated on short axis images and drawn at end-diastole by two independent reviewers on long-axis images. Contours were automatically propagated throughout the cardiac cycle. Global and regional peak systolic strain were compared across observers, field strengths, temporal resolutions, and examinations. Inter-observer agreement was excellent (ICC > 0.87, p < 0.01). Inter-examination variability was low, ranging from 1.7 (1.0-2.4)% to 2.5 (1.9-3.1)%, except for radial strain: 9.2 (7.6-10.5)%. Most global and regional strain values were not significantly different across field strengths and temporal resolutions (p > 0.05). Normal global peak systolic strain values with HDA were -25.0 (-24.0 to -26.1)% (LV circumferential), 60.5 (55.3 to 65.6)% (LV radial), -22.3 (-20.5 to - 24.0)% (LV longitudinal), and -26.0 (-23.8 to -28.2)% (RV longitudinal). HDA prototype software enabled efficient and consistent quantification of myocardial strain from conventional bSSFP cine CMR data, demonstrating clinical feasibility.

Left Atrial and Left Atrial Appendage 4D Blood Flow Dynamics in Atrial Fibrillation.

BACKGROUND: Atrial 4D flow magnetic resonance imaging was used for the characterization of left atrial (LA) and left atrial appendage (LAA) flow dynamics in patients with atrial fibrillation (AF). METHODS AND RESULTS: 4D flow magnetic resonance imaging measured in vivo 3D blood flow velocities in 60 AF patients and 15 controls. Anatomic maps of LA and LAA stasis and velocity were calculated to quantify atrial peak velocity, mean velocity, and stasis (velocities <0.1 m/s). In a substudy with 30 AF patients, 4D flow metrics were compared with Doppler transesophageal echocardiography. For all 15 controls, LAA mean and peak velocities were consistently lower (by 21%/12%; P<0.001) while LAA stasis was higher (by 58%; P<0.001) compared with the LA. In contrast, lower LAA velocity and increased LAA stasis were only found in a fraction (38 of 60) of AF patients. In AF patients, increased CHA2DS2-VASc score was associated with significantly (P<0.043) reduced LA velocities and elevated stasis. There was a heterogeneous expression of atrial flow dynamics, and 25% to 68% of AF patients demonstrated flow in the normal range: 25%/68% for LA/LAA stasis and 38%/60% for LA/LAA peak velocities. Transesophageal echocardiography velocities modestly but significantly (P<0.05) correlated with 4D flow-based LA velocities (r=0.41) and stasis (r=-0.39). CONCLUSIONS: AF resulted in overall impaired but individually variable flow dynamics in both the LA and LAA. AF patients demonstrated atrial flow in the normal range, despite elevated CHA2DS2-VASc score.

Altered aortic shape in bicuspid aortic valve relatives influences blood flow patterns.

AIMS: Bicuspid aortic valve (BAV) is known to exhibit familial inheritance and is associated with aortopathy and altered aortic haemodynamics. However, it remains unclear whether BAV-related aortopathy can be inherited independently of valve morphology. METHODS AND RESULTS: Four-dimensional flow magnetic resonance imaging for the in vivo assessment of thoracic aortic 3D blood flow was performed in 24 BAV relatives with trileaflet aortic valves (age = 40 ± 14 years) and 15 healthy controls (age = 37 ± 10 years). Data analysis included aortic dimensions, shape (round/gothic/cubic), and 3D blood flow characteristics (semi-quantitative vortex/helix grading and peak velocities). Cubic and gothic aortic shapes were markedly more prevalent in BAV relatives compared with controls (38 vs. 7%). Ascending aorta (AAo) vortex flow in BAV relatives was significantly increased compared with controls (grading = 1.5 ± 1.0 vs. 0.6 ± 0.9, P = 0.015). Aortic haemodynamics were influenced by aortic shape: peak velocities were reduced for gothic aortas vs. round aortas (P = 0.003); vortex flow was increased for cubic aortas in the AAo (P < 0.001) and aortic arch (P = 0.004); vortex and helix flows were elevated for gothic aortas in the AAo and descending aorta (P = 0.003, P = 0.029). Logistic regression demonstrated significant associations of shape with severity of vortex flow in AAo (P < 0.001) and aortic arch (P = 0.016) in BAV relatives. CONCLUSION: BAV relatives expressed altered aortic shape and increased vortex flow despite the absence of valvular disease or aortic dilatation. These data suggest a heritable component of BAV-related aortopathy affecting aortic shape and aberrant blood flow, independent of valve morphology.

Right ventricular assessment at cardiac MRI: initial clinical experience utilizing an IS-SENSE reconstruction.

Cardiac MR is considered the gold standard in assessing RV function. The purpose of this study is to evaluate the clinical utility of an investigational iterative reconstruction algorithm in the quantitative assessment of RV function. This technique has the potential to improve the clinical utility of CMR in the evaluation of RV pathologies, particularly in patients with dyspnea, by shortening acquisition times without adversely influencing imaging performance. Segmented cine images were acquired on 9 healthy volunteers and 29 patients without documented RV pathologies using conventional GRAPPA acquisition with factor 2 acceleration (GRAPPA 2), a spatio-temporal TSENSE acquisition with factor 4 acceleration (TSENSE 4), and iteratively reconstructed Sparse SENSE acquisition with factor 4 acceleration (IS-SENSE 4). 14 subjects were re-analyzed and intraclass correlation coefficients (ICC) were calculated and Bland-Altman plots generated to assess agreement. Two independent reviewers qualitatively scored images. Comparison of acquisition techniques was performed using univariate analysis of variance (ANOVA). Differences in RV EF, BSA-indexed ESV (ESVi), BSA-indexed EDV (EDVi), and BSA-indexed SV (SVi) were shown to be statistically insignificant via ANOVA testing. R(2) values for linear regression of TSENSE 4 and IS-SENSE 4 versus GRAPPA 2 were 0.34 and 0.72 for RV-EF, and 0.61 and 0.76 for RV-EDVi. ICC values for intraobserver and interobserver quantification yielded excellent agreement, and Bland-Altman plots assessing agreement were generated as well. Qualitative review yielded small, but statistically significant differences in image quality and noise between TSENSE 4 and IS-SENSE 4. All three techniques were rated nearly artifact free. Segmented imaging acquisitions with IS-SENSE reconstruction and an acceleration factor of 4 accurately and reliably quantitates RV systolic function parameters, while maintaining image quality. TSENSE-4 accelerated acquisitions showed poorer correlation to standard imaging, and inferior interobserver and intraobserver agreement. IS-SENSE has the potential to shorten cine acquisition times by 50 %, improving imaging options in patients with intermittent arrhythmias or difficulties with breath holding.

Highly accelerated cardiac MRI using iterative SENSE reconstruction: initial clinical experience.

To evaluate the qualitative and quantitative performance of an accelerated cardiovascular MRI (CMR) protocol that features iterative SENSE reconstruction and spatio-temporal L1-regularization (IS SENSE). Twenty consecutively recruited patients and 9 healthy volunteers were included. 2D steady state free precession cine images including 3-chamber, 4-chamber, and short axis slices were acquired using standard parallel imaging (GRAPPA, acceleration factor = 2), spatio-temporal undersampled TSENSE (acceleration factor = 4), and IS SENSE techniques (acceleration factor = 4). Acquisition times, quantitative cardiac functional parameters, wall motion abnormalities (WMA), and qualitative performance (scale: 1-poor to 5-excellent for overall image quality, noise, and artifact) were compared. Breath-hold times for IS SENSE (3.0 ± 0.6 s) and TSENSE (3.3 ± 0.6) were both reduced relative to GRAPPA (8.4 ± 1.7 s, p < 0.001). No difference in quantitative cardiac function was present between the three techniques (p = 0.89 for ejection fraction). GRAPPA and IS SENSE had similar image quality (4.7 ± 0.4 vs. 4.5 ± 0.6, p = 0.09) while, both techniques were superior to TSENSE (quality: 4.1 ± 0.7, p < 0.001). GRAPPA WMA agreement with IS SENSE was good (κ > 0.60, p < 0.001), while agreement with TSENSE was poor (κ < 0.40, p < 0.001). IS SENSE is a viable clinical CMR acceleration approach to reduce acquisition times while maintaining satisfactory qualitative and quantitative performance.

Assessment of left and right atrial 3D hemodynamics in patients with atrial fibrillation: a 4D flow MRI study.

Atrial fibrillation (AF) is associated with embolic stroke due to thrombus formation in the left atrium (LA). Based on the relationship of atrial stasis to thromboembolism and the marked disparity in pulmonary versus systemic thromboembolism in AF, we tested the hypothesis that flow velocity distributions in the left (LA) versus right atrium (RA) in patients with would demonstrate increased stasis. Whole heart 4D flow MRI was performed in 62 AF patients (n = 33 in sinus rhythm during imaging, n = 29 with persistent AF) and 8 controls for the assessment of in vivo atrial 3D blood flow. 3D segmentation of the LA and RA geometry and normalized velocity histograms assessed atrial velocity distribution and stasis (% of atrial velocities <0.2 m/s). Atrial hemodynamics were similar for RA and LA and significantly correlated (mean velocity: r = 0.64; stasis: r = 0.55, p < 0.001). RA and LA mean and median velocities were lower in AF patients by 15-33 % and stasis was elevated by 11-19 % compared to controls. There was high inter-individual variability in LA/RA mean velocity ratio (range 0.5-1.8) and LA/RA stasis ratio (range 0.7-1.7). Patients with a history of AF and in sinus rhythm showed most pronounced differences in atrial flow (reduced mean velocities, higher stasis in the LA). While there is no systematic difference in LA versus RA flow velocity profiles, high variability was noted. Further delineation of patient specific factors and/or regional atrial effects on the LA and RA flow velocity profiles, as well as other factors such as differences in procoagulant factors, may explain the more prevalent systemic versus pulmonary thromboembolism in patients with AF.

Left Atrial 4-Dimensional Flow Magnetic Resonance Imaging: Stasis and Velocity Mapping in Patients With Atrial Fibrillation.

OBJECTIVES: Left atrial (LA) 4-dimensional flow magnetic resonance imaging (MRI) was used to derive anatomic maps of LA stasis, peak velocity, and time-to-peak (TTP) velocity in patients with atrial fibrillation (AF) and to identify relationships between LA flow with LA volume and patient characteristics. MATERIALS AND METHODS: Four-dimensional flow MRI for the in vivo assessment of time-resolved 3-dimensional LA blood flow velocities was performed in 111 subjects: 42 patients with a history of AF and in sinus rhythm (AF-sinus), 39 patients with persistent AF (AF-afib), 10 young healthy volunteers (HVs), and 20 age-appropriate controls (CTRL). Data analysis included the 3-dimensional segmentation of the LA and the calculation of LA stasis, peak velocity, and TTP maps. Regional LA flow dynamics were quantified by calculating mean stasis, peak velocity, and TTP in the LA center region and the region adjacent to the LA wall. RESULTS: A sensitivity analysis identified thresholds for global LA stasis (<0.1 m/s) and peak velocity (top 5% LA velocities), which detected significant differences between AF patients and controls for global LA stasis (HV, 25% ± 5%; CTRL, 29% ± 10%; AF-sinus, 41% ± 13%; AF-afib, 52% ± 17%) and peak velocity (HV, 0.43 ± 0.02 m/s; CTRL, 0.37 ± 0.04 m/s; AF-sinus, 0.33 ± 0.05 m/s; AF-afib, 0.30 ± 0.05 m/s). Regional analysis revealed significantly increased stasis at both LA center and wall for AF patients compared with age-appropriate controls (29%-84% difference, P < 0.006) and for AF-afib versus AF-sinus patients (22%-30% difference, P < 0.004). In addition, stasis close to the LA wall was significantly elevated (P < 0.001) compared with the LA center for all subject groups. Multiple regressions revealed significant (RAdj = 0.45-0.50, P < 0.001) relationships between impaired global LA flow (reduced velocity and increased stasis) with age (|ß| = 0.27-0.50, P < 0.002) and LA volume (|ß| = 0.26-0.50, P < 0.003). CONCLUSIONS: Atrial 4-dimensional flow MRI detected changes in global and regional LA flow dynamics associated with AF, age, and LA volume. Longitudinal studies are needed to test the diagnostic value of LA flow metrics as potential risk factors for thromboembolic events.

Three-dimensional left atrial blood flow characteristics in patients with atrial fibrillation assessed by 4D flow CMR.

AIMS: To apply 4D flow cardiac magnetic resonance (CMR) for the volumetric measurement of 3D left atrial (LA) blood flow to evaluate its potential to detect altered LA flow in patients with atrial fibrillation (AF) and to investigate associations of changes in systolic and diastolic LA flow with the current clinical risk score (CHA2DS2-VASc) used for the assessment of thromboembolic risk in AF. METHODS AND RESULTS: 4D flow CMR was performed in 40 patients with a history of AF (in sinus rhythm during CMR scan, age = 61 ± 11 years), 20 age-appropriate controls (59 ± 7 years), and 10 young healthy volunteers (24 ± 2 years) to measure in vivo time-resolved 3D LA blood flow. LA velocities were characterized with respect to atrial function and timing by calculating normalized LA flow velocity histograms during ventricular systole, early diastole, mid-late diastole, and the entire cardiac cycle. Mean, median, and peak LA velocity steadily decreased when comparing young volunteers, age-appropriate controls, and AF patients by 10-44% and 8-26% for early diastole and the entire cardiac cycle, respectively (P < 0.01 for all comparisons except median velocity for young vs. older volunteers and peak velocity for older volunteers and AF patients). There were moderate but significant inverse relationships between increased CHA2DS2-VASc score and reduced mean LA velocity (early diastole: r = -0.37, P < 0.001; entire RR-interval: r = -0.33, P = 0.005), median LA velocity (r = -0.33, P = 0.003; r = -0.25, P = 0.017), and peak velocity (r = -0.36, P = 0.001; r = -0.45, P < 0.001). LA flow indices also correlated significantly with age and LA volume (R2 = 0.44-0.62, P < 0.001), but not with left ventricular ejection fraction. CONCLUSION: Left atrial 4D flow CMR demonstrated significantly reduced LA blood flow velocities in patients with AF. Further study is needed to determine whether these measures can improve upon the CHA2DS2-VASc score for stroke risk prediction and enhance individual decisions on anticoagulation in patients with AF.

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.

Thoracic aorta 3D hemodynamics in pediatric and young adult patients with bicuspid aortic valve.

BACKGROUND: To evaluate the 3D hemodynamics in the thoracic aorta of pediatric and young adult bicuspid aortic valve (BAV) patients. METHODS: 4D flow MRI was performed in 30 pediatric and young adult BAV patients (age: 13.9 ± 4.4 (range: [3.4, 20.7]) years old, M:F = 17:13) as part of this Institutional Review Board-approved study. Nomogram-based aortic root Z-scores were calculated to assess aortic dilatation and degree of aortic stenosis (AS) severity was assessed on MRI. Data analysis included calculation of time-averaged systolic 3D wall shear stress (WSSsys ) along the entire aorta wall, and regional quantification of maximum and mean WSSsys and peak systolic velocity (velsys ) in the ascending aorta (AAo), arch, and descending aorta (DAo). The 4D flow MRI AAo velsys was also compared with echocardiography peak velocity measurements. RESULTS: There was a positive correlation with both mean and max AAo WSSsys and peak AAo velsys (mean: r = 0.84, P < 0.001, max: r = 0.94, P < 0.001) and AS (mean: rS = 0.43, P = 0.02, max: rS = 0.70, P < 0.001). AAo peak velocity was significantly higher when measured with echo compared with 4D flow MRI (2.1 ± 0.98 m/s versus 1.27 ± 0.49 m/s, P < 0.001). CONCLUSION: In pediatric and young adult patients with BAV, AS and peak ascending aorta velocity are associated with increased AAo WSS, while aortic dilation, age, and body surface area do not significantly impact AAo hemodynamics. Prospective studies are required to establish the role of WSS as a risk-stratification tool in these patients.

Characterization of abnormal wall shear stress using 4D flow MRI in human bicuspid aortopathy.

There exists considerable controversy surrounding the timing and extent of aortic resection for patients with BAV disease. Since abnormal wall shear stress (WSS) is potentially associated with tissue remodeling in BAV-related aortopathy, we propose a methodology that creates patient-specific 'heat maps' of abnormal WSS, based on 4D flow MRI. The heat maps were created by detecting outlier measurements from a volumetric 3D map of ensemble-averaged WSS in healthy controls. 4D flow MRI was performed in 13 BAV patients, referred for aortic resection and 10 age-matched controls. Systolic WSS was calculated from this data, and an ensemble-average and standard deviation (SD) WSS map of the controls was created. Regions of the individual WSS maps of the BAV patients that showed a higher WSS than the mean + 1.96SD of the ensemble-average control WSS map were highlighted. Elevated WSS was found on the greater ascending aorta (35% ± 15 of the surface area), which correlated significantly with peak systolic velocity (R (2) = 0.5, p = 0.01) and showed good agreement with the resected aortic regions. This novel approach to characterize regional aortic WSS may allow clinicians to gain unique insights regarding the heterogeneous expression of aortopathy and may be leveraged to guide patient-specific resection strategies for aorta repair.

Three-dimensional hemodynamics in intracranial aneurysms: influence of size and morphology.

PURPOSE: To use four-dimensional (4D)-flow MRI for the comprehensive in vivo analysis of hemodynamics and its relationship to size and morphology of different intracranial aneurysms (IA). We hypothesize that different IA groups, defined by size and morphology, exhibit different velocity fields, wall shear stress, and vorticity. MATERIALS AND METHODS: The 4D-flow MRI (spatial resolution = 0.99-1.8 × 0.78-1.46 × 1.2-1.4 mm(3) , temporal resolution = 44-48 ms) was performed in 19 IAs (18 patients, age = 55.4 ± 13.8 years) with saccular (n = 16) and fusiform (n = 3) morphology and different sizes ranging from small (n = 8; largest dimension = 6.2 ± 0.4 mm) to large and giant (n = 11; 25 ± 7 mm). Analysis included quantification of volumetric spatial-temporal velocity distribution, vorticity, and wall shear stress (WSS) along the aneurysm's 3D surface. RESULTS: The 4D-flow MRI revealed distinct hemodynamic patterns for large/giant saccular aneurysms (Group 1), small saccular aneurysms (Group 2), and large/giant fusiform aneurysms (Group 3). Saccular IA (Groups 1, 2) demonstrated significantly higher peak velocities (P < 0.002) and WSS (P < 0.001) compared with fusiform aneurysms. Although intra-aneurysmal 3D velocity distributions were similar for Group 1 and 2, vorticity and WSS was significantly (P < 0.001) different (increased in Group 1 by 54%) indicating a relationship between IA size and hemodynamics. Group 3 showed reduced velocities (P < 0.001) and WSS (P < 0.001). CONCLUSION: The 4D-flow MRI demonstrated the influence of lesion size and morphology on aneurysm hemodynamics suggesting the potential of 4D-flow MRI to assist in the classification of individual aneurysms.

Discovery of an allosteric mechanism for the regulation of HCV NS3 protein function.

Here we report a highly conserved new binding site located at the interface between the protease and helicase domains of the hepatitis C virus (HCV) NS3 protein. Using a chemical lead, identified by fragment screening and structure-guided design, we demonstrate that this site has a regulatory function on the protease activity via an allosteric mechanism. We propose that compounds binding at this allosteric site inhibit the function of the NS3 protein by stabilizing an inactive conformation and thus represent a new class of direct-acting antiviral agents.

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.

Fragment-based drug discovery applied to Hsp90. Discovery of two lead series with high ligand efficiency.

Inhibitors of the chaperone Hsp90 are potentially useful as chemotherapeutic agents in cancer. This paper describes an application of fragment screening to Hsp90 using a combination of NMR and high throughput X-ray crystallography. The screening identified an aminopyrimidine with affinity in the high micromolar range and subsequent structure-based design allowed its optimization into a low nanomolar series with good ligand efficiency. A phenolic chemotype was also identified in fragment screening and was found to bind with affinity close to 1 mM. This fragment was optimized using structure based design into a resorcinol lead which has subnanomolar affinity for Hsp90, excellent cell potency, and good ligand efficiency. This fragment to lead campaign improved affinity for Hsp90 by over 1,000,000-fold with the addition of only six heavy atoms. The companion paper (DOI: 10.1021/jm100060b) describes how the resorcinol lead was optimized into a compound that is now in clinical trials for the treatment of cancer.

Discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1-ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (AT13387), a novel inhibitor of the molecular chaperone Hsp90 by fragment based drug design.

Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are currently generating significant interest in clinical development as potential treatments for cancer. In a preceding publication (DOI: 10.1021/jm100059d ) we describe Astex's approach to screening fragments against Hsp90 and the subsequent optimization of two hits into leads with inhibitory activities in the low nanomolar range. This paper describes the structure guided optimization of the 2,4-dihydroxybenzamide lead molecule 1 and details some of the drug discovery strategies employed in the identification of AT13387 (35), which has progressed through preclinical development and is currently being tested in man.