ZNL0325, a Pyrazolopyrimidine-Based Covalent Probe, Demonstrates an Alternative Binding Mode for Kinases.

The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket. ZNL0325 and its analogues feature an acrylamide side chain at the C3 position which is capable of forming a covalent bond with multiple kinases that possess a cysteine at the αD-1 position including BTK, EGFR, BLK, and JAK3. These findings suggest that the ability to form a covalent bond can override the preferred noncovalent binding conformation of the heterocyclic core and provides an opportunity to create structurally distinct covalent kinase inhibitors.

Investigation of scatter radiation intensities in the cardiac catheter laboratory: novel versus traditional shielding solutions.

A manufacturer has released a novel shielding solution (NSS): Rampart M1128 and claimed that the personal protective equipment (PPE) can be removed. This study investigates the scatter intensities with the NSS or the traditional shielding solutions (TSS) including the ceiling-suspended screen and the tableside lead drape. Isodose maps were generated by two series of measurements with an anthropomorphic phantom using NSS and TSS. Three survey meters were positioned at different heights to measure the scatter intensities at the eye, chest, and pelvic levels. Additional measurements were made at the primary and secondary operators? locations to evaluate the scatter intensities with different clinical projections. For the main operator positions, the isodose maps showed that NSS could result in a scatter dose that reduced by 80% to 95% compared to the same positions with TSS at the eye and chest levels. The corresponding result at the pelvic level was a reduction of 50%. These reductions should be compared to the additional protection by PPE: up to 80% reduction from lead eyeglasses and up to 95% from protective garments. Considering both operators at clinically relevant LAO projections, NSS resulted in scatter dose that was 80% to 96%, 76% to 96% and 25% to 60% lower than those of the TSS at eye, chest and pelvis levels. The protection of NSS is comparable with that of TSS alongside PPE at the eye but not at the chest and the pelvic levels under the setup of coronary angiography.

Borrowing Transcriptional Kinases to Activate Apoptosis.

Protein kinases are disease drivers whose therapeutic targeting traditionally centers on inhibition of enzymatic activity. Here chemically induced proximity is leveraged to convert kinase inhibitors into context-specific activators of therapeutic genes. Bivalent molecules that link ligands of the transcription factor B-cell lymphoma 6 (BCL6) to ATP-competitive inhibitors of cyclin-dependent kinases (CDKs) were developed to re-localize CDK to BCL6-bound loci on chromatin and direct phosphorylation of RNA Pol II. The resulting BCL6-target proapoptotic gene expression translated into killing of diffuse large B-cell lymphoma (DLBCL) cells at 72 h with EC50s of 0.9 - 10 nM and highly specific ablation of the BCL6-regulated germinal center response in mice. The molecules exhibited 10,000-fold lower cytotoxicity in normal lymphocytes and are well tolerated in mice. Genomic and proteomic evidence corroborated a gain-of-function mechanism where, instead of global enzyme inhibition, a fraction of total kinase activity is borrowed and re-localized to BCL6-bound loci. The strategy demonstrates how kinase inhibitors can be used to context-specifically activate transcription, accessing new therapeutic space.

Targeted kinase degradation via the KLHDC2 ubiquitin E3 ligase.

Chemically induced protein degradation is a powerful strategy for perturbing cellular biochemistry. The predominant mechanism of action for protein degrader drugs involves an induced proximity between the cellular ubiquitin-conjugation machinery and a target. Unlike traditional small molecule enzyme inhibition, targeted protein degradation can clear an undesired protein from cells. We demonstrate here the use of peptide ligands for Kelch-like homology domain-containing protein 2 (KLHDC2), a substrate adapter protein and member of the cullin-2 (CUL2) ubiquitin ligase complex, for targeted protein degradation. Peptide-based bivalent compounds that can induce proximity between KLHDC2 and target proteins cause degradation of the targeted factors. The cellular activity of these compounds depends on KLHDC2 binding. This work demonstrates the utility of KLHDC2 for targeted protein degradation and exemplifies a strategy for the rational design of peptide-based ligands useful for this purpose.

Trial of a Novel Radiation Shielding Device to Protect Staff in the Cardiac Catheter Laboratory.

Continuous exposure to low-level scattered radiation to staff performing cardiac angiography and intervention is of concern. A novel shielding solution (NSS) (Rampart IC M1128) has the potential to provide greater shielding for staff present at the table-side. This study aimed to investigate the effectiveness of the NSS compared with a traditional shielding solution (TSS) in a randomized controlled trial that enrolled 100 patients who underwent cardiac angiography and/or intervention which were randomized to the NSS or TSS. Baseline patient characteristics and radiation dose data were collected. Staff who were scrubbed at the table-side wore 5 real-time dosimeters on the head, collar, waist, ankle, and under the apron. The median primary operator radiation dose was significantly lower (p <0.001) for all dosimeter locations with the NSS when compared with the TSS, being reduced by 86%, 80.0%, 100%, and 50.0% for the head, collar, waist, and leg respectively. Median under-apron dose was 0.0 µSv for both NSS and TSS. Median second operator dose was reduced by 100%, 100%, and 100% for the head, collar, and waist respectively (p <0.001). Median NSS and TSS dose at the ankle and under apron was 0.0 µSv. Median scrub nurse dose was reduced by 50% and 100% for the head and collar respectively (p <0.001). Median NSS and TSS dose at the waist, ankle, and under apron was 0.0 µSv. In conclusion, the NSS tested in this study demonstrates a significant decrease in radiation dose to operators and scrub nurses when compared with traditional radiation protection measures.

Structure-Based Design of Y-Shaped Covalent TEAD Inhibitors.

Transcriptional enhanced associate domain (TEAD) proteins together with their transcriptional coactivator yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) are important transcription factors and cofactors that regulate gene expression in the Hippo pathway. In mammals, the TEAD families have four homologues: TEAD1 (TEF-1), TEAD2 (TEF-4), TEAD3 (TEF-5), and TEAD4 (TEF-3). Aberrant expression and hyperactivation of TEAD/YAP signaling have been implicated in a variety of malignancies. Recently, TEADs were recognized as being palmitoylated in cells, and the lipophilic palmitate pocket has been successfully targeted by both covalent and noncovalent ligands. In this report, we present the medicinal chemistry effort to develop MYF-03-176 (compound 22) as a selective, cysteine-covalent TEAD inhibitor. MYF-03-176 (compound 22) significantly inhibits TEAD-regulated gene expression and proliferation of the cell lines with TEAD dependence including those derived from mesothelioma and liposarcoma.

Development of a Covalent Inhibitor of c-Jun N-Terminal Protein Kinase (JNK) 2/3 with Selectivity over JNK1.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1-JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher Kinact/KI compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.

Covalent disruptor of YAP-TEAD association suppresses defective Hippo signaling.

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

Calculations of dose point kernels of 64 Cu in different media with PENELOPE Monte Carlo code.

PURPOSE: The unique decay properties of copper-64 (64 Cu) has made it a radionuclide of interest in theragnostic applications of nuclear medicine. This study aims to calculate the dose point kernels (DPKs) of 64 Cu in various media with PENELOPE Monte Carlo code. METHODS: Monte Carlo simulations were performed using PENELOPE code (version 2014). To calculate DPKs, the simulation comprised an isotropic point radiation source positioned at the origin of a spherical object of radius 50 cm. The absorbed dose along the radial direction outwards from the point source were scored with a resolution of 20 µm. Validations were firstly performed by calculating the DPKs of monoenergetic electrons and photons in water and the results were compared against the literature values. The continuous energy spectra of the beta minus and positron emissions from 64 Cu were numerically modeled and used as inputs to the simulation. DPKs of 64 Cu were calculated in water, soft tissue, lung tissue, and cortical bone, including all emissions types. RESULTS: The simulations have been successfully validated against literature values. The largest deviations have been observed with 10 keV monoenergetic electrons with the average and maximum dose difference of -1.01% and -10.56%. The modeled energy spectra closely compared with the average energies from Brookhaven Laboratory National Nuclear Data Centre and the combined spectral shapes from the RAdiation Dose Assessment Resource (RADAR). The DPKs of 64 Cu demonstrated different radial dose deposition in different media owing to the different physical density and effective atomic number. CONCLUSIONS: The DPKs of 64 Cu have been calculated with Monte Carlo simulations in four different media. They will be useful to study the dosimetric properties of 64 Cu-labeled radiopharmaceuticals and perform therapeutic dose planning.

Dosimetric impact of breast density in breast tomosynthesis: A comparative study.

A radiation dose survey has been undertaken involving 256 patients to investigate the dosimetric impact of breast tomosynthesis screening by employing different breast densities estimated by the Dance model, 50-50 breast model, and patient-specific density software: Volpara. Mean glandular dose (MGD) based on the Dance model provided the most realistic dose estimate with an average difference of -3.3 ± 4.8% from the patient-specific estimation. Average differences of -8.2 ± 6.5% and -7.3 ± 4.7% were observed for the 50-50 breast model and console MGD, respectively. We conclude that the Dance model should be used for dose calculations in radiation dose surveys and establishing diagnostic reference levels (DRL).

A method to obtain correct standard uptake values in Pinnacle treatment planning system for target volume delineation.

Standardized uptake value (SUV) is an advanced tool for quantitative tumor identification and metabolic target volume delineation (TVD) in diagnostic and therapeutic settings. It is thus important to establish a quality assured process to maintain the traceability of data correctly by positron emission tomography (PET) systems. Patient administration of 18fluoro-deoxy-glucose is increasingly delivered by automated infusion systems (AISs). Whenever AIS is used, its accuracy and traceability measurement need verification. In addition, it was observed that the unreproducible SUV displayed in PET and the treatment planning system (TPS) may cause grave concerns for radiation oncologists for TVD. This concern may complicate the correlation of TVD on PET and TPS and their clinical reporting. The SUV traceability was established from the PET system to AIS. Its accuracy was verified by cross-referencing to the reference dose calibrator traceable to a primary standard. The SUV values were converted in TPS using the in-house "clinical tool" to be identical as in PET, to allow radiation oncologists to use SUV confidently. The outcome of this study enables the clinical groups to rely on the correct SUV values displayed on the TPS and to improve the quality of care for patients in clinical procedures.

Investigation of the clinical performance of a novel solid-state diagnostic dosimeter.

This study investigated the clinical performance of a novel solid-state diagnostic dosimeter, the RaySafe Xi transparent detector, by comparing its performance to a reference-class ionization chamber. Firstly a comparison of dosimeter response "free-in-air" with standard beam qualities was made, followed by an investigation into its relative transparency in an X-ray field and angular sensitivity dependence. The second part of the study looked at the overall performance of the transparent detector under scatter conditions with a number of beam qualities, including standard beam and those hardened by copper (Cu) filtration of thickness up to 0.9 mm, as would be encountered in the equipment testing of fluoroscopy systems. Overall, the transparent detector has demonstrated equivalent measurement properties to the ionization chamber under standard conditions and provided similar X-ray attenuation as reflected by the nearly identical radiographic parameters selected for both dosimeters by the automatic dose rate control (ADRC) system. Yet, it also possessed an asymmetric angular response which respectively under- and overestimated the dose contribution from the rear and lateral directions by the same amount of 50%. The transparent detector provided comparable dose reading of ± 3% to the ionization chamber with standard beam qualities and backscatter radiation present. These results were in good agreement with those of free-in-air measurement, indicating that the angular under- and overresponse might potentially compensate one another for accurate measurement. However, for identical Cu filtered beam qualities and setups, the transparent detector on average overresponded by 5.4% across the useful tube voltage range. In conclusion, the transparent detector, with its novel design, is essentially equivalent, within a 5% tolerance, to an ionization chamber, except in situations where beams hardened with Cu filtration are used with backscatter radiation present requiring larger uncertainty error estimations.