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The existence of multiple centrosomes in some cancer cells can lead to cell death through the formation of multipolar mitotic spindles and consequent aberrant cell division. Many cancer cells rely on HSET (KIFC1) to cluster the extra centrosomes into two groups to mimic the bipolar spindle formation of non-centrosome-amplified cells and ensure their survival. Here, we report the discovery of a novel 2-(3-benzamidopropanamido)thiazole-5-carboxylate with micromolar in vitro inhibition of HSET (KIFC1) through high-throughput screening and its progression to ATP-competitive compounds with nanomolar biochemical potency and high selectivity against the opposing mitotic kinesin Eg5. Induction of the multipolar phenotype was shown in centrosome-amplified human cancer cells treated with these inhibitors. In addition, a suitable linker position was identified to allow the synthesis of both fluorescent- and trans -cyclooctene (TCO)-tagged probes, which demonstrated direct compound binding to the HSET protein and confirmed target engagement in cells, through a click-chemistry approach.

Purpose: To develop a Breast Imaging Reporting and Data System (BI-RADS) breast density deep learning (DL) model in a multisite setting for synthetic two-dimensional mammographic (SM) images derived from digital breast tomosynthesis examinations by using full-field digital mammographic (FFDM) images and limited SM data., Materials and Methods: A DL model was trained to predict BI-RADS breast density by using FFDM images acquired from 2008 to 2017 (site 1: 57 492 patients, 187 627 examinations, 750 752 images) for this retrospective study. The FFDM model was evaluated by using SM datasets from two institutions (site 1: 3842 patients, 3866 examinations, 14 472 images, acquired from 2016 to 2017; site 2: 7557 patients, 16 283 examinations, 63 973 images, 2015 to 2019). Each of the three datasets were then split into training, validation, and test. Adaptation methods were investigated to improve performance on the SM datasets, and the effect of dataset size on each adaptation method was considered. Statistical significance was assessed by using CIs, which were estimated by bootstrapping., Results: Without adaptation, the model demonstrated substantial agreement with the original reporting radiologists for all three datasets (site 1 FFDM: linearly weighted Cohen κ [κ w ] = 0.75 [95% CI: 0.74, 0.76]; site 1 SM: κ w = 0.71 [95% CI: 0.64, 0.78]; site 2 SM: κ w = 0.72 [95% CI: 0.70, 0.75]). With adaptation, performance improved for site 2 (site 1: κ w = 0.72 [95% CI: 0.66, 0.79], 0.71 vs 0.72, P = .80; site 2: κ w = 0.79 [95% CI: 0.76, 0.81], 0.72 vs 0.79, P < .001) by using only 500 SM images from that site., Conclusion: A BI-RADS breast density DL model demonstrated strong performance on FFDM and SM images from two institutions without training on SM images and improved by using few SM images. Supplemental material is available for this article. Published under a CC BY 4.0 license., Competing Interests: Disclosures of Conflicts of Interest: T.P.M. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed money paid to author for employment from Whiterabbit; disclosed stock/stock options in Whiterabbit; disclosed money to author’s institution for patents related to Whiterabbit. Other relationships: disclosed no relevant relationships. S.S. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed payment to author for employment from Whiterabbit. Other relationships: disclosed no relevant relationships. B.M. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed money paid to author for employment from Whiterabbit; disclosed stock/stock options in Whiterabbit; disclosed money to author’s institution for patents related to Whiterabbit. Other relationships: disclosed no relevant relationships. J.S. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed money paid to author for board membership from Whiterabbit; disclosed employment from Whiterabbit; disclosed stock/stock options from Whiterabbit. Other relationships: disclosed no relevant relationships. M.P.S. disclosed no relevant relationships. S.P. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed money paid to author for employment from Whiterabbit. Other relationships: disclosed no relevant relationships. A.L. disclosed no relevant relationships. D.M. disclosed no relevant relationships. J.G. disclosed no relevant relationships. R.M.H. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed payment to author for employment from Whiterabbit; disclosed stock/stock options from Whiterabbit. Other relationships: disclosed no relevant relationships. N.G. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed payment to author for employment from Whiterabbit; disclosed stock/stock options from Whiterabbit. Other relationships: disclosed no relevant relationships. D.S. disclosed no relevant relationships. S.M.M. Activities related to the present article: disclosed money to author’s institution from Whiterabbit as a grant. Activities not related to the present article: disclosed no relevant relationships. Other relationships: disclosed no relevant relationships. S.C.M. disclosed no relevant relationships. R.L.W. Activities related to the present article: disclosed grant to author’s institution from Whiterabbit. Activities not related to the present article: disclosed possibility of royalties if the FDA approves a related product; disclosed stock options for Washington University from Whiterabbit. Other relationships: disclosed no relevant relationships., (2020 by the Radiological Society of North America, Inc.)

A series of imidazo[1,2- b]pyridazin-8-amine kinase inhibitors were discovered to allosterically inhibit the endoribonuclease function of the dual kinase-endoribonuclease inositol-requiring enzyme 1α (IRE1α), a key component of the unfolded protein response in mammalian cells and a potential drug target in multiple human diseases. Inhibitor optimization gave compounds with high kinome selectivity that prevented endoplasmic reticulum stress-induced IRE1α oligomerization and phosphorylation, and inhibited endoribonuclease activity in human cells. X-ray crystallography showed the inhibitors to bind to a previously unreported and unusually disordered conformation of the IRE1α kinase domain that would be incompatible with back-to-back dimerization of the IRE1α protein and activation of the endoribonuclease function. These findings increase the repertoire of known IRE1α protein conformations and can guide the discovery of highly selective ligands for the IRE1α kinase site that allosterically inhibit the endoribonuclease.

Accurate estimation of the initial pressure distribution in photoacoustic computed tomography (PACT) depends on knowledge of the sound speed distribution. However, the sound speed distribution is typically unknown. Further, the initial pressure and sound speed distributions cannot both, in general, be stably recovered from PACT measurements alone. In this work, a joint reconstruction (JR) method for the initial pressure distribution and a low-dimensional parameterized model of the sound speed distribution is proposed. By employing a priori information about the structure of the sound speed distribution, both the initial pressure and sound speed can be accurately recovered. The JR problem is solved by use of a proximal optimization method that allows constraints and non-smooth regularization functions for the initial pressure distribution. The gradients of the cost function with respect to the initial pressure and sound speed distributions are calculated by use of an adjoint state method that has the same per-iteration computational cost as calculating the gradient with respect to the initial pressure distribution alone. This approach is evaluated through 2D computer-simulation studies for a small animal imaging model and by application to experimental in vivo measurements of a mouse.

The initial pressure and speed of sound (SOS) distributions cannot both be stably recovered from photoacoustic computed tomography (PACT) measurements alone. Adjunct ultrasound computed tomography (USCT) measurements can be employed to estimate the SOS distribution. Under the conventional image reconstruction approach for combined PACT/USCT systems, the SOS is estimated from the USCT measurements alone and the initial pressure is estimated from the PACT measurements by use of the previously estimated SOS. This approach ignores the acoustic information in the PACT measurements and may require many USCT measurements to accurately reconstruct the SOS. In this work, a joint reconstruction method where the SOS and initial pressure distributions are simultaneously estimated from combined PACT/USCT measurements is proposed. This approach allows accurate estimation of both the initial pressure distribution and the SOS distribution while requiring few USCT measurements.

Ultrasound computed tomography (USCT) holds great promise for breast cancer screening. Waveform inversion-based image reconstruction methods account for higher order diffraction effects and can produce high-resolution USCT images, but are computationally demanding. Recently, a source encoding technique has been combined with stochastic gradient descent (SGD) to greatly reduce image reconstruction times. However, this method bundles the stochastic data fidelity term with the deterministic regularization term. This limitation can be overcome by replacing SGD with a structured optimization method, such as the regularized dual averaging method, that exploits knowledge of the composition of the cost function. In this paper, the dual averaging method is combined with source encoding techniques to improve the effectiveness of regularization while maintaining the reduced reconstruction times afforded by source encoding. It is demonstrated that each iteration can be decomposed into a gradient descent step based on the data fidelity term and a proximal update step corresponding to the regularization term. Furthermore, the regularization term is never explicitly differentiated, allowing nonsmooth regularization penalties to be naturally incorporated. The wave equation is solved by the use of a time-domain method. The effectiveness of this approach is demonstrated through computer simulation and experimental studies. The results suggest that the dual averaging method can produce images with less noise and comparable resolution to those obtained by the use of SGD.

Photoacoustic computed tomography (PACT) and ultrasound computed tomography (USCT) are emerging modalities for breast imaging. As in all emerging imaging technologies, computer-simulation studies play a critically important role in developing and optimizing the designs of hardware and image reconstruction methods for PACT and USCT. Using computer-simulations, the parameters of an imaging system can be systematically and comprehensively explored in a way that is generally not possible through experimentation. When conducting such studies, numerical phantoms are employed to represent the physical properties of the patient or object to-be-imaged that influence the measured image data. It is highly desirable to utilize numerical phantoms that are realistic, especially when task-based measures of image quality are to be utilized to guide system design. However, most reported computer-simulation studies of PACT and USCT breast imaging employ simple numerical phantoms that oversimplify the complex anatomical structures in the human female breast. We develop and implement a methodology for generating anatomically realistic numerical breast phantoms from clinical contrast-enhanced magnetic resonance imaging data. The phantoms will depict vascular structures and the volumetric distribution of different tissue types in the breast. By assigning optical and acoustic parameters to different tissue structures, both optical and acoustic breast phantoms will be established for use in PACT and USCT studies.

Photoacoustic computed tomography (PACT) is an emerging computed imaging modality that exploits optical contrast and ultrasonic detection principles to form images of the absorbed optical energy density within tissue. If the object possesses spatially variant acoustic properties that are unaccounted for by the reconstruction method, the estimated image can contain distortions. While reconstruction methods have recently been developed to compensate for this effect, they generally require the object’s acoustic properties to be known a priori. To circumvent the need for detailed information regarding an object’s acoustic properties, we previously proposed a half-time reconstruction method for PACT. A half-time reconstruction method estimates the PACT image from a data set that has been temporally truncated to exclude the data components that have been strongly aberrated. However, this method can be improved upon when the approximate sizes and locations of isolated heterogeneous structures, such as bones or gas pockets, are known. To address this, we investigate PACT reconstruction methods that are based on a variable data truncation (VDT) approach. The VDT approach represents a generalization of the half-time approach, in which the degree of temporal truncation for each measurement is determined by the distance between the corresponding ultrasonic transducer location and the nearest known bone or gas void location. Computer-simulated and experimental data are employed to demonstrate the effectiveness of the approach in mitigating artifacts due to acoustic heterogeneities.

Photoacoustic computed tomography (PACT) is an emerging imaging modality that exploits optical contrast and ultrasonic detection principles to form images of the photoacoustically induced initial pressure distribution within tissue. The PACT reconstruction problem corresponds to an inverse source problem in which the initial pressure distribution is recovered from measurements of the radiated wavefield. A major challenge in transcranial PACT brain imaging is compensation for aberrations in the measured data due to the presence of the skull. Ultrasonic waves undergo absorption, scattering and longitudinal-to-shear wave mode conversion as they propagate through the skull. To properly account for these effects, a wave-equation-based inversion method should be employed that can model the heterogeneous elastic properties of the skull. In this work, a forward model based on a finite-difference time-domain discretization of the three-dimensional elastic wave equation is established and a procedure for computing the corresponding adjoint of the forward operator is presented. Massively parallel implementations of these operators employing multiple graphics processing units (GPUs) are also developed. The developed numerical framework is validated and investigated in computer19 simulation and experimental phantom studies whose designs are motivated by transcranial PACT applications.

The heat shock protein 70s (HSP70s) are molecular chaperones implicated in many cancers and of significant interest as targets for novel cancer therapies. Several HSP70 inhibitors have been reported, but because the majority have poor physicochemical properties and for many the exact mode of action is poorly understood, more detailed mechanistic and structural insight into ligand-binding to HSP70s is urgently needed. Here we describe the first comprehensive fragment-based inhibitor exploration of an HSP70 enzyme, which yielded an amino-quinazoline fragment that was elaborated to a novel ATP binding site ligand with different physicochemical properties to known adenosine-based HSP70 inhibitors. Crystal structures of amino-quinazoline ligands bound to the different conformational states of the HSP70 nucleotide binding domain highlighted the challenges of a fragment-based approach when applied to this particular flexible enzyme class with an ATP-binding site that changes shape and size during its catalytic cycle. In these studies we showed that Ser275 is a key residue in the selective binding of ATP. Additionally, the structural data revealed a potential functional role for the ATP ribose moiety in priming the protein for the formation of the ATP-bound pre-hydrolysis complex by influencing the conformation of one of the phosphate binding loops., Competing Interests: The authors are current or former employees of The Institute of Cancer Research, which has a commercial interest in the development of molecular chaperone inhibitors.

Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.

CCT245737 is the first orally active, clinical development candidate CHK1 inhibitor to be described. The IC50 was 1.4 nM against CHK1 enzyme and it exhibited>1,000-fold selectivity against CHK2 and CDK1. CCT245737 potently inhibited cellular CHK1 activity (IC50 30-220 nM) and enhanced gemcitabine and SN38 cytotoxicity in multiple human tumor cell lines and human tumor xenograft models. Mouse oral bioavailability was complete (100%) with extensive tumor exposure. Genotoxic-induced CHK1 activity (pS296 CHK1) and cell cycle arrest (pY15 CDK1) were inhibited both in vitro and in human tumor xenografts by CCT245737, causing increased DNA damage and apoptosis. Uniquely, we show CCT245737 enhanced gemcitabine antitumor activity to a greater degree than for higher doses of either agent alone, without increasing toxicity, indicating a true therapeutic advantage for this combination. Furthermore, development of a novel ELISA assay for pS296 CHK1 autophosphorylation, allowed the quantitative measurement of target inhibition in a RAS mutant human tumor xenograft of NSCLC at efficacious doses of CCT245737. Finally, CCT245737 also showed significant single-agent activity against a MYC-driven mouse model of B-cell lymphoma. In conclusion, CCT245737 is a new CHK1 inhibitor clinical development candidate scheduled for a first in man Phase I clinical trial, that will use the novel pS296 CHK1 ELISA to monitor target inhibition.

Photoacoustic computed tomography (PACT) is an emerging computed imaging modality that exploits optical contrast and ultrasonic detection principles to form images of the absorbed optical energy density within tissue. When the imaging system employs conventional piezoelectric ultrasonic transducers, the ideal photoacoustic (PA) signals are degraded by the transducers' acousto-electric impulse responses (EIRs) during the measurement process. If unaccounted for, this can degrade the accuracy of the reconstructed image. In principle, the effect of the EIRs on the measured PA signals can be ameliorated via deconvolution; images can be reconstructed subsequently by application of a reconstruction method that assumes an idealized EIR. Alternatively, the effect of the EIR can be incorporated into an imaging model and implicitly compensated for during reconstruction. In either case, the efficacy of the correction can be limited by errors in the assumed EIRs. In this work, a joint optimization approach to PACT image reconstruction is proposed for mitigating errors in reconstructed images that are caused by use of an inaccurate EIR. The method exploits the bi-linear nature of the imaging model and seeks to refine the measured EIR during the process of reconstructing the sought-after absorbed optical energy density. Computer-simulation and experimental studies are conducted to investigate the numerical properties of the method and demonstrate its value for mitigating image distortions and enhancing the visibility of fine structures.

Peripheral neuropathy is a common neurological problem that affects millions of people worldwide. Diagnosis and treatment of this condition are often hindered by the difficulties in making objective, noninvasive measurements of nerve fibers. Photoacoustic microscopy (PAM) has the ability to obtain high resolution, specific images of peripheral nerves without exogenous contrast. We demonstrated the first proof-of-concept imaging of peripheral nerves using PAM. As validated by both standard histology and photoacoustic spectroscopy, the origin of photoacoustic signals is myelin, the primary source of lipids in the nerves. An extracted sciatic nerve sandwiched between two layers of chicken tissue was imaged by PAM to mimic the in vivo case. Ordered fibrous structures inside the nerve, caused by the bundles of myelin-coated axons, could be observed clearly. With further technical improvements, PAM can potentially be applied to monitor and diagnose peripheral neuropathies.

Introduction: Checkpoint kinase (CHK) inhibitors offer the promise of enhancing the effectiveness of widely prescribed cancer chemotherapies and radiotherapy by inhibiting the DNA damage response, as well as the potential for single agent efficacy., Areas Covered: This article surveys structural insights into the checkpoint kinases CHK1 and CHK2 that have been exploited to enhance the selectivity and potency of small molecule inhibitors. Furthermore, the authors review the use of mechanistic cellular assays to guide the optimisation of inhibitors. Finally, the authors discuss the status of the current clinical candidates and emerging new clinical contexts for CHK1 and CHK2 inhibitors, including the prospects for single agent efficacy., Expert Opinion: Protein-bound water molecules play key roles in structural features that can be targeted to gain high selectivity for either enzyme. The results of early phase clinical trials of checkpoint inhibitors have been mixed, but significant progress has been made in testing the combination of CHK1 inhibitors with genotoxic chemotherapy. Second-generation CHK1 inhibitors are likely to benefit from increased selectivity and oral bioavailability. While the optimum therapeutic context for CHK2 inhibition remains unclear, the emergence of single agent preclinical efficacy for CHK1 inhibitors in specific tumour types exhibiting constitutive replication stress represents exciting progress in exploring the therapeutic potential of these agents.

Inhibitors of checkpoint kinase 1 (CHK1) are of current interest as potential antitumor agents, but the most advanced inhibitor series reported to date are not orally bioavailable. A novel series of potent and orally bioavailable 3-alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitrile CHK1 inhibitors was generated by hybridization of two lead scaffolds derived from fragment-based drug design and optimized for CHK1 potency and high selectivity using a cell-based assay cascade. Efficient in vivo pharmacokinetic assessment was used to identify compounds with prolonged exposure following oral dosing. The optimized compound (CCT244747) was a potent and highly selective CHK1 inhibitor, which modulated the DNA damage response pathway in human tumor xenografts and showed antitumor activity in combination with genotoxic chemotherapies and as a single agent.

Purpose: Many tumors exhibit defective cell-cycle checkpoint control and increased replicative stress. CHK1 is critically involved in the DNA damage response and maintenance of replication fork stability. We have therefore discovered a novel potent, highly selective, orally active ATP-competitive CHK1 inhibitor, CCT244747, and present its preclinical pharmacology and therapeutic activity., Experimental Design: Cellular CHK1 activity was assessed using an ELISA assay, and cytotoxicity a SRB assay. Biomarker modulation was measured using immunoblotting, and cell-cycle effects by flow cytometry analysis. Single-agent oral CCT244747 antitumor activity was evaluated in a MYCN-driven transgenic mouse model of neuroblastoma by MRI and in genotoxic combinations in human tumor xenografts by growth delay., Results: CCT244747 inhibited cellular CHK1 activity (IC(50) 29-170 nmol/L), significantly enhanced the cytotoxicity of several anticancer drugs, and abrogated drug-induced S and G(2) arrest in multiple tumor cell lines. Biomarkers of CHK1 (pS296 CHK1) activity and cell-cycle inactivity (pY15 CDK1) were induced by genotoxics and inhibited by CCT244747 both in vitro and in vivo, producing enhanced DNA damage and apoptosis. Active tumor concentrations of CCT244747 were obtained following oral administration. The antitumor activity of both gemcitabine and irinotecan were significantly enhanced by CCT244747 in several human tumor xenografts, giving concomitant biomarker modulation indicative of CHK1 inhibition. CCT244747 also showed marked antitumor activity as a single agent in a MYCN-driven neuroblastoma., Conclusion: CCT244747 represents the first structural disclosure of a highly selective, orally active CHK1 inhibitor and warrants further evaluation alone or combined with genotoxic anticancer therapies., (©2012 AACR)

With possibilities for radiation terrorism and intensified concerns about nuclear accidents since the recent Fukushima Daiichi event, the potential exposure of large numbers of individuals to radiation that could lead to acute clinical effects has become a major concern. For the medical community to cope with such an event and avoid overwhelming the medical care system, it is essential to identify not only individuals who have received clinically significant exposures and need medical intervention but also those who do not need treatment. The ability of electron paramagnetic resonance to measure radiation-induced paramagnetic species, which persist in certain tissues (e.g., teeth, fingernails, toenails, bone, and hair), has led to this technique becoming a prominent method for screening significantly exposed individuals. Although the technical requirements needed to develop this method for effective application in a radiation event are daunting, remarkable progress has been made. In collaboration with General Electric and through funding committed by the Biomedical Advanced Research and Development Authority, electron paramagnetic resonance tooth dosimetry of the upper incisors is being developed to become a Food and Drug Administration-approved and manufacturable device designed to carry out triage for a threshold dose of 2 Gy. Significant progress has also been made in the development of electron paramagnetic resonance nail dosimetry based on measurements of nails in situ under point-of-care conditions, and in the near future this may become a second field-ready technique. Based on recent progress in measurements of nail clippings, it is anticipated that this technique may be implementable at remotely located laboratories to provide additional information when the measurements of dose on-site need to be supplemented. The authors conclude that electron paramagnetic resonance dosimetry is likely to be a useful part of triage for a large-scale radiation incident.

Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.

In order to meet the potential need for emergency large-scale retrospective radiation biodosimetry following an accident or attack, we have developed instrumentation and methodology for in vivo electron paramagnetic resonance spectroscopy to quantify concentrations of radiation-induced radicals within intact teeth. This technique has several very desirable characteristics for triage, including independence from confounding biologic factors, a non-invasive measurement procedure, the capability to make measurements at any time after the event, suitability for use by non-expert operators at the site of an event, and the ability to provide immediate estimates of individual doses. Throughout development there has been a particular focus on the need for a deployable system, including instrumental requirements for transport and field use, the need for high throughput, and use by minimally trained operators.Numerous measurements have been performed using this system in clinical and other non-laboratory settings, including in vivo measurements with unexposed populations as well as patients undergoing radiation therapies. The collection and analyses of sets of three serially-acquired spectra with independent placements of the resonator, in a data collection process lasting approximately five minutes, provides dose estimates with standard errors of prediction of approximately 1 Gy. As an example, measurements were performed on incisor teeth of subjects who had either received no irradiation or 2 Gy total body irradiation for prior bone marrow transplantation; this exercise provided a direct and challenging test of our capability to identify subjects who would be in need of acute medical care.

Rapid and accurate retrospective dosimetry is of critical importance and strategic value for the emergency medical response to a large-scale radiological/nuclear event. One technique that has the potential for rapid and accurate dosimetry measurements is electron paramagnetic resonance (EPR) spectroscopy of relatively stable radiation-induced signals (RIS) in fingernails and toenails. Two approaches are being developed for EPR nail dosimetry. In the approach using ex vivo measurements on nail clippings, accurate estimation of the dose-dependent amplitude of the RIS is complicated by the presence of mechanically-induced signals (MIS) that are generated during the nail clipping. Recent developments in ex vivo nail dosimetry, including a thorough characterization of the MIS and an appreciation of the role of hydration and the development of effective analytic techniques, have led to improvements in the accuracy and precision of this approach. An in vivo nail dosimetry approach is also very promising, as it eliminates the problems of MIS from the clipping and it has the potential to be an effective and efficient approach for field deployment. Two types of EPR resonators are being developed for in vivo measurements of fingernails and toenails.

Purpose: The ability to estimate individual exposures to radiation following a large attack or incident has been identified as a necessity for rational and effective emergency medical response. In vivo electron paramagnetic resonance (EPR) spectroscopy of tooth enamel has been developed to meet this need., Materials and Methods: A novel transportable EPR spectrometer, developed to facilitate tooth dosimetry in an emergency response setting, was used to measure upper incisors in a model system, in unirradiated subjects, and in patients who had received total body doses of 2 Gy., Results: A linear dose response was observed in the model system. A statistically significant increase in the intensity of the radiation-induced EPR signal was observed in irradiated versus unirradiated subjects, with an estimated standard error of dose prediction of 0.9 ± 0.3 Gy., Conclusions: These results demonstrate the current ability of in vivo EPR tooth dosimetry to distinguish between subjects who have not been irradiated and those who have received exposures that place them at risk for acute radiation syndrome. Procedural and technical developments to further increase the precision of dose estimation and ensure reliable operation in the emergency setting are underway. With these developments EPR tooth dosimetry is likely to be a valuable resource for triage following potential radiation exposure of a large population.

A lead-like kinase inhibitor screening library containing new 3-aminopyrazolopyridinones and closely related compounds was designed that contained hydrogen-bond donor-acceptor motifs and substitution vectors inspired by the natural product kinase inhibitor indirubin. The solubility of the 3-aminopyrazolopyridinone scaffold was more than 1000-fold greater than that of indirubin itself, and solubility was enhanced by reduction of the proportion of lipophilic aryl substituents or the introduction of basic groups. Several components of the library showed kinase inhibitory activity. A subset of diaryl-substituted analogues preferentially inhibited tyrosine kinases with low micromolar activity and good ligand efficiency, and showed cellular antiproliferative activity. The evaluation of the library shows that new, non-natural compounds with relevant biological activity and improved physicochemical properties can be generated from the natural product indirubin, providing compounds that may be useful for kinase inhibitor drug discovery., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A range of 3,6-di(hetero)arylimidazo[1,2-a]pyrazine ATP-competitive inhibitors of CHK1 were developed by scaffold hopping from a weakly active screening hit. Efficient synthetic routes for parallel synthesis were developed to prepare analogues with improved potency and ligand efficiency against CHK1. Kinase profiling showed that the imidazo[1,2-a]pyrazines could inhibit other kinases, including CHK2 and ABL, with equivalent or better potency depending on the pendant substitution. These 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines appear to represent a general kinase inhibitor scaffold., (2010 Elsevier Ltd. All rights reserved.)

Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells.

We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K(d) = 1.7 nmol/L) and proliferation of human tumor cells with GI(50) values of approximately 2 to 40 nmol/L, inducing G(1)-G(2) arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI(50). This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1alpha, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro, WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI50 averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by approximately 50%.

Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors.

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.

The structure and electronic properties of a series of biologically active 2-nitrothiophenes (1) have been calculated using both semi-empirical and ab initio molecular orbital methods. Multi-linear regression analysis suggests that there is a reasonable correlation between the experimental activity of the derivatives against either Escherichia coli or Micrococcus luteus and calculated properties such as the HOMO energies, the total atomic charges and ring angles at the heterocyclic sulfur atom, but there is no correlation with the calculated solvation energies or dipole moments. The presence or absence of an additional nitro group at the 3-position of the ring also has a significant effect on the activity. From the derived QSAR equations, the 2-chloro- or 2-bromo-3,5-dinitrothiophenes (1a and 1c) are predicted to show the highest activity against both bacteria, while 2-nitrothiophene (1n) is predicted to be the least active, in line with the experimental results.

The biological activity of nineteen substituted thiophenes (3) have been assessed by evaluating the minimum inhibitory concentration required to inhibit the growth of E. coli, M. luteus and A. niger. The series displays a wide range of activities with 2-chloro-3,5-dinitrothiophene (3a) or 2-bromo-3,5-dinitrothiophene (3c) showing the highest activity against all three organisms, while the simplest compound of the series, 2-nitrothiophene (3s) shows the smallest activity in each case. The mode of action of 3a and 3c is thought to involve nucleophilic attack by intracellular thiols at the 2-position of the heterocyclic ring leading to displacement of halogen, but other active derivatives, such as 2,4-dinitrothiophene (3h) and 5-nitrothiophene-2-carbaldehyde (3d) which have no displaceable halogen or leaving group are thought to act by forming Meisenheimer complexes.

High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 microM) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical properties. Some initial structure-activity relationships are discussed, as well as the crystal structure of CCT018159 bound to Hsp90.