Endoplasmic reticulum protein BI-1 regulates Ca²âº-mediated bioenergetics to promote autophagy.

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²âº mediated by inositol triphosphate receptors (IP3Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP3R-dependent manner. By reducing steady-state levels of ER Ca²âº via IP3Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1's ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca²âº signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.

Effectiveness of a Shoulder Exercise Program in Division I Collegiate Baseball Players During the Fall Season.

BACKGROUND: Deficits in shoulder range of motion (ROM) and strength are associated with risk of arm injury in baseball players. PURPOSE: The purpose of this study was to assess the effectiveness of a standardized exercise program, during the fall season, on shoulder ROM and rotational strength in collegiate baseball players. STUDY DESIGN: Prospective cohort study. METHODS: Passive shoulder internal rotation (IR), external rotation (ER), and horizontal adduction ROM were measured with an inclinometer. Shoulder IR and ER strength was assessed using a hand-held dynamometer and normalized to body weight. Players performed a program of shoulder stretching and strengthening exercises, three times/week for one month and then one time/week for two months. Paired sample t-tests compared pre-intervention to post-intervention outcome measures. RESULTS: Division I baseball players (n=43; 19.6±1.2years, 185.8±5.5cm, 90.5±7.0kg) volunteered. From pre- to post-intervention, there were increases in horizontal adduction ROM in the throwing (Mean Difference (MD)=6.1°, 95%CI=3.7,8.5; p<0.001) and non-throwing arm (MD=8.0°, 95%CI=5.6,10.3; p<0.001), and a decrease in non-throwing arm ER ROM (MD=2.8°, 95%CI= 0.2,5.5; p=0.039). The ER ROM surplus (throwing - non-throwing) increased (MD=5.6°, 95%CI= 1.1,10.2; p=0.016). Throwing arm (MD=1.3%BW, 95%CI=0.5-2.1, p=0.003) and non-throwing arm (MD=1.2%BW, 95%CI=0.4,2.0; p=0.004) ER strength decreased. A notable, but non-significant increase in IR strength on the throwing arm (MD=1.6%BW, 95%CI=0.1,3.0; p=0.055) and decrease on the non-throwing arm (MD=1.2%BW, 95%CI=0.0,2.4; p=0.055) occurred. Additionally, throwing arm ER:IR strength ratio (MD=0.16, 95%CI=0.08,0.25; p<0.001) also decreased. CONCLUSION: Changes in shoulder horizontal adduction ROM, IR strength and relative ER surplus on the throwing arm were noted at the end of the season. The lack of change in IR and ER ROM and may be related to the lack of deficits at the start of the fall season. LEVEL OF EVIDENCE: 2.

Epidemiology and Outcomes of Maxillofacial Injuries in NCAA Division I Athletes Participating in 13 Sports.

Background: Maxillofacial injuries account for an estimated 11% of National Collegiate Athletic Association (NCAA) sport-related injuries and occur at a rate of 0.2-1.5 injuries per 1000 athletic events/exposures. Purpose: The purpose of this study was to report the epidemiology, treatment, and outcomes of maxillofacial injuries in NCAA Division I athletes participating in 13 sports. It was hypothesized that the rate of maxillofacial injuries would be greater than previously reported in national registry studies. Study Design: Descriptive epidemiology study. Methods: A single-institution registry was utilized to retrieve the maxillofacial injuries and surgical procedures recorded over 4 athletic seasons, for the years 2015 through 2019, across 13 NCAA Division I sports. The incidence of injuries per sport was reported as the number per 1000 athlete-exposure (AE) hours. The time lost from participation and time to complete injury resolution per sport were reported as the mean and range. Results: A total of 193 maxillofacial injuries occurred over 4 seasons. The overall incidence of maxillofacial injuries was 2.06 injuries per 1000 AE hours. The injury incidence for male and female athletes was 1.92 and 2.43 injuries per 1000 AE hours, respectively. Men's basketball (8.30 injuries per 1000 AE hours) and men's water polo (8.15 injuries per 1000 AE hours) had the highest rates of all sports. Overall, 20 athletes (10.4%) required surgery. The mean time to resolution across all sports was 33.3 days (range, 0-336 days) per injury. The mean time lost across all sports was 17.1 days (range, 0-336 days) per injury. Conclusion: At a single NCAA Division I institution, maxillofacial injuries occurred at a higher rate than previously thought and could lead to significant time lost from sport participation. Basketball players were at the highest risk of this injury. Across all sports, male athletes took longer to return to sport after a maxillofacial injury compared to female athletes, but the latter required more time to fully recover. Maxillofacial injuries may require surgical treatment, and their prevention is critical.

Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics.

Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Analysis of the 2.5 kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many ß-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a ß-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes through the NF-κB signaling pathway. The NF-κB binding site at -272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.

NONOPERATIVE TREATMENT FOR INJURIES TO THE IN-SEASON THROWING SHOULDER: A CURRENT CONCEPTS REVIEW WITH CLINICAL COMMENTARY.

Repetitive overhead throwing generates tremendous demands on the shoulder joint of the overhead athlete. Clinicians, therapists, and medical staff are charged with optimizing a throwing athlete's shoulder mobility and stability to maximize performance and prevent injury. Modifiable risk factors such as strength asymmetry, glenohumeral range of motion deficits, and scapulothoracic joint abnormalities contribute to the overhead athlete's predisposition to shoulder injury. Most shoulder injuries in the overhead thrower can be successfully treated nonoperatively to allow in-season return to sport. The optimal rehabilitation program must be based on an accurate evaluation of historical and physical information as well as diagnostic imaging. Return to play decisions should be individualized and should weigh subjective assessments along with objective measurements of range of motion, strength, and function. The purpose of this clinical commentary is to summarize the current literature regarding the nonoperative treatment options for these common injuries, and to present a treatment plan to safely return these athletes to the field of play. Level of evidence: 5.

Factores asociados a dolor lumbar en trabajadores sanitarios de un hospital de referencia del Perú

Introducción: El dolor lumbar es una condición inevitable en todo el personal del rubro de la salud, con llevando desde malestar físico hasta una incapacidad funcional del individuo. Objetivo: Determinar los factores asociados a dolor lumbar entre los trabajadores sanitarios en un hospital de referencia del Perú. Metodología: El presente estudio es observacional, analítico, de corte transversal, temporalmente prospectivo, con muestreo no probabilístico. La población estuvo conformada por trabajadores sanitarios del Hospital Santa Rosa de Pueblo Libre durante el periodo de junio a diciembre del año 2022. Resultados: El análisis multivariado determinó que el ser hombre (OR: 2.818, p valor: 0.017), tener sobrepeso (OR:1.782, p valor: 0.013), demanda laboral alta (OR: 4.750, p valor: 0.026), realizar actividad física (OR: 3.610, p valor: 0.031) y tener antecedentes de trauma lumbar (OR: 2.423, p valor: 0.034), fueron factores estadísticamente significativos que se asociaron al dolor lumbar. Discusión: Se pudo observar que, los factores asociados a dolor lumbar fueron el sexo masculino, el sobrepeso, la demanda laboral alta, el realizar actividad física y el antecedente de trauma lumbar. Conocer estas variables permitirá realizar esquemas y charlas preventivas para afrontar esta recurrente patología.

Introduction: Low back pain is an unavoidable condition in all health personnel, ranging from physical discomfort to functional disability of the individual. Objective: To determine the factors associated with low back pain among health workers at a reference hospital in Peru. Methods: This study is observational, analytical, cross-sectional, temporally prospective, with non-probabilistic sampling. The population was made up of health workers from the Hospital Santa Rosa de Pueblo Libre during the period from June to December of the year 2022. Results: The multivariate analysis determined that being a man (OR: 2.818, p value: 0.017), being overweight (OR:1.782, p value: 0.013), high labor demand (OR: 4.750, p value: 0.026), performing physical (OR: 3.610, p value: 0.031) and having a history of low back trauma (OR: 2.423, p value: 0.034) were statistically significant factors associated with low back pain. Discussion: It was possible to observe that the factors associated with low back pain were the male sex, being overweight, high work demand, physical activity and a history of low back trauma. Knowing these variables will make it possible to carry out preventive schemes and talks to deal with this recurring pathology.

[Percutaneous nephrolithotomy (PCNL): Conditions for a safe performance.] / Nefrolitotricia percutanea (NLP): condiciones de una ejecución segura.

OBJECTIVE: To describe the PCNL technique addressing those conditions that enable us to perform the procedure with maximum patient's safety without losing focus on the objective of lithiasis resolution. METHODS: Based on our experience, accumulated with time, and the constant update of the endourological techniques we treat those issues in relation to PCNL from the efficiency, efficacy and effectiveness perspective. To consider them involves the successful performance of the technique, with complications prevention and taking cost-efficiency into account. The first one is in close connection to selection of the most adequate technique for each case, training and team work to achieve excellence. With meticulous preoperative preparation and procedure performance, on every single step of the operation, we will ensure an effective technique, avoiding complications. Efficiency comes from the two first and involves, in addition to perform a successful and safe procedure, that the procedure should be cost effective from the management point of view. CONCLUSIONS: PCNL offers the possibility of endoscopic extraction of renal lithiasis, regardless of its volume, density or site, but its success, safety and efficiency are closely linked to the possibility of establishment of an appropriate tract that enables manipulation and total extraction of the calculus.

Factores asociados a bajo conocimiento de bioseguridad en internos de medicina en una universidad del Perú en el contexto de la pandemia COVID-19

Introducción: El derecho a prácticas pre-profesionales amparado bajo la Ley Peruana permite que el estudiante de último año de la carrera de medicina humana comience a participar en procedimientos e intervenciones asistenciales de salud bajo la guía de un médico tutor. Objetivo: determinar los factores asociados a bajo conocimiento en bioseguridad en internos de medicina de una universidad del Perú en el contexto de la pandemia covid-19. Metodología: se realizó un diseño observacional, prolectivo y transversal. La muestra estuvo comprendida por 336 internos de medicina humana, los cuales se seleccionaron por muestreo probabilístico aleatorio simple. Se empleó un instrumento válido y confiable para determinar el nivel de conocimiento en bioseguridad. Se trabajó con un modelo de regresión de binario logarítmico bivariado y múltiple para el cálculo de la razón de prevalencia cruda y ajustada con sus respectivos intervalos de confianza al 95% para los factores asociados al nivel de conocimiento en bioseguridad. Resultados: se encontraron niveles de conocimiento alto, medio y bajo del 44,6%, 26,8% y 28 ,6% respectivamente. El estado civil casado (RPA 0.418, IC95% 0.181 - 0.968), convivir con personas con riesgo para síntomas graves por covid-19 (RPA 0.472, IC95% 0.344-0.647), realizar el internado medico en un hospital y haber sido diagnosticado por covid-19 en los últimos 12 meses (RPA 0.586, IC95% 0.387-0.887) se asociaron a un nivel de conocimiento bajo bioseguridad por modelo de regresión binaria logarítmica múltiple. Discusión: el estado civil casado, el convivir con personas con riesgo para síntomas graves de covid-19, el realizar el internado medico en un centro hospitalario y el haber sido diagnosticado con covid-19 tienen una relación inversamente proporcional con el conocimiento bajo en bioseguridad en internos de medicina en el contexto de la pandemia covid-19.

Introduction: The right to pre-professional practices protected under Peruvian Law allows the student in the last year of the human medicine career to begin to participate in health care procedures and interventions under the guidance of a tutor. Objective: to determine the factors associated with low knowledge of biosafety in medical interns at a university in Peru in the context of the covid-19 pandemic. Methods: an observational, prolective and cross-sectional design was carried out. The sample consisted of 336 human medicine interns, who were selected by simple random probabilistic sampling. A valid and reliable instrument was used to determine the level of knowledge in biosafety. A bivariate and multiple logarithmic binary regression model was used to calculate the crude and adjusted prevalence ratio with their respective 95% confidence intervals for the factors associated with the level of knowledge in biosafety. Results: high, medium and low levels of knowledge of 44.6%, 26.8% and 28.6% respectively were found. Married marital status (RPA 0.418, 95% CI 0.181 - 0.968), living with people at risk for severe symptoms from covid-19 (RPA 0.472, 95% CI 0.344-0.647), undergoing medical internship in a hospital, and having been diagnosed by covid-19 in the last 12 months (RPA 0.586, 95% CI 0.387-0.887) were associated with a level of knowledge under biosafety by multiple logarithmic binary regression model. Discussion: married marital status, living with people at risk for severe symptoms of covid-19, undergoing medical internship in a hospital center and having been diagnosed with covid-19 have an inversely proportional relationship with low knowledge in biosafety. In medical interns in the context of the covid-19 pandemic.

Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML).

Cardiac glycosides (CGs), inhibitors of Na+/K+-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli.

Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death.

Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent.

Cell-Based High-Throughput Luciferase Reporter Gene Assays for Identifying and Profiling Chemical Modulators of Endoplasmic Reticulum Signaling Protein, IRE1.

Endoplasmic reticulum (ER) stress activates three distinct signal transducers on the ER membrane. Inositol-requiring protein 1 (IRE1), the most conserved signal transducer, plays a key role in ER stress-mediated signaling. During ER stress, IRE1 initiates two discrete signaling cascades: the "adaptive" signaling cascade mediated by the XBP1 pathway and the "alarm" signaling cascade mediated by stress-activated protein kinase pathways. Fine-tuning of the balance between the adaptive and alarm signals contributes significantly to cellular fate under ER stress. Thus, we propose that the design of high-throughput screening (HTS) assays to selectively monitor IRE1 mediated-signaling would be desirable for drug discovery. To this end, we report the generation of stable human neural cell lines and development of cell-based HTS luciferase (Luc) reporter gene assays for the identification of pathway-specific chemical modulators of IRE1. We implemented a cell-based Luc assay using a chimeric CHOP-Gal4 transcription factor in 384-well format for monitoring IRE1 kinase-mediated p38MAPK activation and an unfolded response pathway element (URPE)-Luc cell-based assay in 1536-well format for monitoring IRE1's RNase-mediated activation of XBP1. Chemical library screening was successfully conducted with both the CHOP/Gal4-Luc cells and UPRE-Luc engineered cells. The studies demonstrate the feasibility of using these HTS assays for discovery of pathway-selective modulators of IRE1.

Does tamoxifen use affect prognosis in breast cancer patients who develop endometrial cancer?

OBJECTIVE: The use of tamoxifen to prevent breast cancer and decrease recurrence is not controversial. However, the effect that tamoxifen may have in women with a history of breast cancer in whom endometrial cancer develops is unclear. The purpose of this study was to estimate whether a history of tamoxifen use is a prognostic factor for such patients. METHODS: Between 1990 and 2002, patients seen at The University of Texas M. D. Anderson Cancer Center with a history of breast cancer who developed endometrial cancer were identified. Medical records were reviewed to identify clinical, pathologic, and outcome information. RESULTS: Eighty-nine patients with a history of breast cancer in whom endometrial carcinoma developed were identified. Fifty-two percent (46/89) had a history of tamoxifen use (median duration 48 months; range 2-120 months). There were no significant differences in the clinical or pathologic features between tamoxifen users and nonusers. A history of tamoxifen use was associated with a shorter interval from breast cancer to endometrial cancer diagnosis (77.2 versus 121.3 months for nonusers; P =.01). There was no significant difference in overall survival between tamoxifen users and nonusers (39.2 months versus 48.3 months, P =.27), and there was no difference in endometrial cancer-specific survival duration between tamoxifen users and nonusers (55.7 versus 51.0 months, P =.92). CONCLUSION: Among tamoxifen users, the interval from breast cancer to endometrial cancer diagnosis was significantly shorter than that in nonusers. In this cohort, a history of tamoxifen use was not associated with a worse overall or disease-specific survival.

Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach.

Interleukin-1 receptor-associated kinase-2 (IRAK2) is a critical mediator of endoplasmic reticulum (ER) stress signaling.

Endoplasmic reticulum (ER) stress occurs when unfolded proteins accumulate in the lumen of the organelle, triggering signal transduction events that contribute either to cellular adaptation and recovery or alternatively to cellular dysfunction and death. ER stress has been implicated in numerous diseases. To identify novel modulators of ER stress, we undertook a siRNA library screen of the kinome, revealing Interleukin-1 Receptor-Associated Kinase-2 (IRAK2) as a contributor to unfolded protein response (UPR) signaling and ER stress-induced cell death. Knocking down expression of IRAK2 (but not IRAK1) in cultured mammalian cells suppresses ER stress-induced expression of the pro-apoptotic transcription factor CHOP and activation of stress kinases. Similarly, RNAi-mediated silencing of the IRAK family member Tube (but not Pelle) suppresses activation of stress kinase signaling induced by ER stress in Drosophila cells. The action of IRAK2 maps to the IRE1 pathway, rather than the PERK or ATF6 components of the UPR. Interestingly, ER stress also induces IRAK2 gene expression in an IRE1/XBP1-dependent manner, suggesting a mutually supporting amplification loop involving IRAK2 and IRE1. In vivo, ER stress induces Irak2 expression in mice. Moreover, Irak2 gene knockout mice display defects in ER stress-induced CHOP expression and IRE1 pathway signaling. These findings demonstrate an unexpected linkage of the innate immunity machinery to UPR signaling, revealing IRAK2 as a novel amplifier of the IRE1 pathway.

TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors.

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.

High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1.

Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

Selected approaches for rational drug design and high throughput screening to identify anti-cancer molecules.

Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specificpromoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4.

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA(2)) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z' factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules (n = 1280 for Lopac™ and 2000 for Spectrum™ library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA(2) and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA(2) reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.