Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells.

HSP90 has emerged as an appealing anti-cancer target. However, HSP90 inhibitors (HSP90i) are characterized by limited clinical utility, primarily due to the resistance acquisition via heat shock response (HSR) induction. Understanding the roles of abundantly expressed cytosolic HSP90 isoforms (α and ß) in sustaining malignant cells' growth and the mechanisms of resistance to HSP90i is crucial for exploiting their clinical potential. Utilizing multi-omics approaches, we identified that ablation of the HSP90ß isoform induces the overexpression of HSP90α and extracellular-secreted HSP90α (eHSP90α). Notably, we found that the absence of HSP90α causes downregulation of PTPRC (or CD45) expression and restricts in vivo growth of BCR-ABL1+ leukemia cells. Subsequently, chronic long-term exposure to the clinically advanced HSP90i PU-H71 (Zelavespib) led to copy number gain and mutation (p.S164F) of the HSP90AA1 gene, and HSP90α overexpression. In contrast, acquired resistance toward other tested HSP90i (Tanespimycin and Coumermycin A1) was attained by MDR1 efflux pump overexpression. Remarkably, combined CDK7 and HSP90 inhibition display synergistic activity against therapy-resistant BCR-ABL1+ patient leukemia cells via blocking pro-survival HSR and HSP90α overexpression, providing a novel strategy to avoid the emergence of resistance against treatment with HSP90i alone.

The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin.

BACKGROUND: The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified. METHODS: We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy. RESULTS: We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound. CONCLUSION: Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract.

Solid-Phase Synthesis of Cereblon-Recruiting Selective Histone Deacetylase 6 Degraders (HDAC6 PROTACs) with Antileukemic Activity.

In this work, we utilized the proteolysis targeting chimera (PROTAC) technology to achieve the chemical knock-down of histone deacetylase 6 (HDAC6). Two series of cereblon-recruiting PROTACs were synthesized via a solid-phase parallel synthesis approach, which allowed the rapid preparation of two HDAC6 degrader mini libraries. The PROTACs were either based on an unselective vorinostat-like HDAC ligand or derived from a selective HDAC6 inhibitor. Notably, both PROTAC series demonstrated selective degradation of HDAC6 in leukemia cell lines. The best degraders from each series (denoted A6 and B4) were capable of degrading HDAC6 via ternary complex formation and the ubiquitin-proteasome pathway, with DC50 values of 3.5 and 19.4 nM, respectively. PROTAC A6 demonstrated promising antiproliferative activity via inducing apoptosis in myeloid leukemia cell lines. These findings highlight the potential of this series of degraders as effective pharmacological tools for the targeted degradation of HDAC6.

The mycotoxin viriditoxin induces leukemia- and lymphoma-specific apoptosis by targeting mitochondrial metabolism.

Inhibition of the mitochondrial metabolism offers a promising therapeutic approach for the treatment of cancer. Here, we identify the mycotoxin viriditoxin (VDT), derived from the endophytic fungus Cladosporium cladosporioides, as an interesting candidate for leukemia and lymphoma treatment. VDT displayed a high cytotoxic potential and rapid kinetics of caspase activation in Jurkat leukemia and Ramos lymphoma cells in contrast to solid tumor cells that were affected to a much lesser extent. Most remarkably, human hematopoietic stem and progenitor cells and peripheral blood mononuclear cells derived from healthy donors were profoundly resilient to VDT-induced cytotoxicity. Likewise, the colony-forming capacity was affected only at very high concentrations, which provides a therapeutic window for cancer treatment. Intriguingly, VDT could directly activate the mitochondrial apoptosis pathway in leukemia cells in the presence of antiapoptotic Bcl-2 proteins. The mitochondrial toxicity of VDT was further confirmed by inhibition of mitochondrial respiration, breakdown of the mitochondrial membrane potential (ΔΨm), the release of mitochondrial cytochrome c, generation of reactive oxygen species (ROS), processing of the dynamin-like GTPase OPA1 and subsequent fission of mitochondria. Thus, VDT-mediated targeting of mitochondrial oxidative phosphorylation (OXPHOS) might represent a promising therapeutic approach for the treatment of leukemia and lymphoma without affecting hematopoietic stem and progenitor cells.

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

Laying an Early Foundation: Lifestyle Medicine Pre-Professional Education (LMPP) Member Interest Group.

Just as lifestyle medicine is the necessary foundation for true health care reform, lifestyle medicine competencies should be the foundation for health education. Although lifestyle medicine education may benefit a health professional at any stage in their education or career, evidence-based undergraduate lifestyle medicine education for future health professionals shifts the perspective of health and health care delivery. Educating health preprofessionals in associate, bachelor's, master's, and other preprofessional healthcare training programs is of paramount importance due to the interdisciplinary nature of lifestyle medicine. To accomplish this, American College of Lifestyle Medicine (ACLM) members can work collaboratively through committees, projects, and working groups-becoming leadership champions of change. An ACLM Pre-Professional Member Interest Group (LMPP) was created in 2018. LMPP has been working to build a national collaborative effort to amass, create, and distribute resources for educators in this pre-professional arena. Educating college students planning to become professionals outside the medical sphere, for example, lawyers, business people, artists, and engineers, will also benefit the field by introducing the power of nutrition, exercise, sleep, social connection, and stress resiliency during this formative state of career development. Pre-professional educational programs provide learners the opportunity to personally experience the power of lifestyle medicine.

Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins.

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH-CC with biotin used in conjunction with streptavidin-horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the Kd values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 µM, 6.0 ± 2.9 µM, and 10.06 ± 2.87 µM, respectively) and found them to be close to previously established Kd values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.

Tool guide for lifestyle behavior change in a cardiovascular risk reduction program.

Lifestyle medicine behaviors such as a healthy diet, physical activity, and tobacco avoidance, are the cornerstone of treatment in many chronic disease conditions, especially those related to the cardiovascular system. In fact, 80% of premature heart disease, stroke, and diabetes may be prevented through modification of these behaviors. The rate-limiting step in cardiovascular disease prevention is the implementation and maintenance of healthy lifestyle behaviors. The purpose of this paper is to provide and discuss a series of tools and strategies that can be used by health care providers to promote health behavior change in their practice.

Phosphonate- and carboxylate-based self-assembled monolayers for organic devices: a theoretical study of surface binding on aluminum oxide with experimental support.

We report a computational study on the chemical bonding of phosphonates and carboxylates to aluminum oxide surfaces and how the binding properties are related to the amount of water in the experimental environment. Two different surface structures were used in the calculations in order to model representative adsorption sites for the phosphonates and carboxylates and to account for the amorphous nature of the hydroxylated AlOx films in experiment. For the phosphonates, we find that the thermodynamically preferred binding mode changes between mono-, bi-, and tridentate depending on the surface structure and the amount of residual water. For the carboxylates, on the other hand, monodentate adsorption is always lower in energy at all experimental conditions. Phosphonates are more strongly bound to aluminum oxide than carboxylates, so that carboxylates can be replaced easily by phosphonates. The theoretical findings are consistent with those obtained in adsorption, desorption, and exchange reactions of n-alkyl phosphonic and carboxylic acids on AlOx surfaces. The results provide an atomistic understanding of the adsorption and help to optimize experimental conditions for self-assembly of organic films on aluminum oxide surfaces.

Reducing the risk of obesity: defining the role of weight loss drugs.

The prevalence of obesity has increased dramatically in the past 20 years. As a public health concern, obesity is associated with a health care resource burden that is quickly approaching that associated with tobacco use. Although lifestyle intervention (diet and exercise) remains the mainstay of treatment of obesity, its effectiveness is limited by poor long-term adherence. Drug therapy has historically been unsuccessful in producing sustained weight loss. Many older weight loss drugs have adverse benefit-to-risk profiles. This review provides an overview of nonpharmacologic interventions for weight loss. The safety and efficacy of older weight loss drugs, as well as current data related to lorcaserin, phentermine/topiramate, and naltrexone-bupropion, are evaluated. Although associated with modest weight loss and some improvement in adverse obesity-related metabolic effects, none of these drugs has been demonstrated to reduce mortality. In addition, the long-term safety of these drugs remains largely unknown. Bariatric surgery is an option for patients with morbid obesity who have failed conventional treatment.

Labeling and enrichment of Arabidopsis thaliana matrix metalloproteases using an active-site directed, marimastat-based photoreactive probe.

Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed a photoreactive probe based on the MMP inhibitor marimastat, conjugated to a 4-azidotetrafluorobenzoyl moiety as photoreactive group and biotin as detection or sorting function. The probe labels At2-MMP, At4-MMP, At5-MMP, and likely other plant MMPs in leaf extracts, as shown by transient At-MMP expression in Nicotiana benthamiana, protein blot, and LC-MS/MS analysis. This MMP probe is a valuable tool to study the post-translational status of MMPs during plant immunity and other MMP-regulated processes.

Profiling of methyltransferases and other S-Adenosyl-L-homocysteine-binding proteins by Capture Compound mass spectrometry.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions. We describe a generic approach based on trifunctional Capture Compounds, in which the initial equilibrium-driven interaction between a small molecule probe and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function of the Capture Compound and the surface of the target protein(s). Subsequently, Capture Compound - protein conjugates are isolated from complex biological mixtures via the sorting function of the Capture Compound. Here, we describe the application of a trifunctional Capture Compound that carries the methyltransferase product inhibitor S-Adenosyl-L -homocysteine as the selectivity function for the isolation of methyltransferases from a complex lysate of Escherichia coli DH5α cells. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free S-Adenosyl-L -homocysteine.

Implementing lifestyle medicine with medication therapy management services to improve patient-centered health care.

OBJECTIVE: To describe a patient-centered medication therapy management (MTM) program that focuses on lifestyle medicine. SETTING: Community pharmacy in Omaha, NE, from August 2008 to September 2010. PRACTICE DESCRIPTION: Traditional MTM services are combined with lifestyle medicine interventions for employees of a self-insured organization who have dyslipidemia, hypertension, and/or diabetes. Program participants meet one-on-one with a pharmacist 12 times during the first year of the program to ensure proper drug therapy and modify lifestyle behaviors (physical activity, nutrition, weight control, sleep, stress, and alcohol and tobacco use) through individualized programming. PRACTICE INNOVATION: Several patient-centered activities have been developed for the program with an emphasis on modifying lifestyle behaviors in conjunction with medications to manage participants' chronic condition. In addition, a new specialty position in health care is being developed (the ambulatist) that focuses on maintaining the ambulatory status of individuals with chronic medical conditions through appropriate drug therapy, lifestyle medicine, and care coordination. MAIN OUTCOME MEASURES: Biometric data collection and participant survey data at baseline and after 12 months. RESULTS: Pilot data for 15 participants showed improvements in all measurements, including blood cholesterol, low-density lipoprotein cholesterol, blood glucose, body weight, physical activity level, fruit and vegetable intake, risk for myocardial infarction, risk for any cardiovascular disease event, self-reported unhealthy days, and qualitative survey data. CONCLUSION: Pharmacists are in an ideal position to implement lifestyle medicine strategies in combination with MTM services to enhance patient-centered health care in a community pharmacy setting.

Transdermal patch drug delivery interactions with exercise.

Transdermal drug delivery systems, such as the transdermal patch, continue to be a popular and convenient way to administer medications. There are currently several medications that use a transdermal patch drug delivery system. This article describes the potential untoward side effects of increased drug absorption through the use of a transdermal patch in individuals who exercise or participate in sporting events. Four studies have been reported that demonstrate a significant increase in the plasma concentration of nitroglycerin when individuals exercise compared with rest. Likewise, several case reports and two studies have been conducted that demonstrate nicotine toxicity and increased plasma nicotine while wearing a nicotine patch in individuals who exercise or participate in sporting events compared with rest. Healthcare providers, trainers and coaches should be aware of proper transdermal patch use, especially while exercising, in order to provide needed information to their respective patients and athletes to avoid potential untoward side effects. Particular caution should be given to individuals who participate in an extreme sporting event of long duration. Further research that includes more medications is needed in this area.

Profiling of methyltransferases and other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS).

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography (1) or Activity Based Protein Profiling (2). Trifunctional Capture Compounds (CCs, Figure 1A) (3) are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-(L)-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" (4) or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules). S-Adenosyl-(L)-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature (5, 6). It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA (7), RNA (8), proteins (9), or small molecules (10). Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A). SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor (11). For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability (12), SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5α of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Synthesis of S-adenosyl-L-homocysteine capture compounds for selective photoinduced isolation of methyltransferases.

Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.

Lifestyle modifications for patients with hypertension.

OBJECTIVES: To review the lifestyle modification components listed in the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7) from the National Heart, Lung, and Blood Institute (NHLBI) and discuss how the guidelines can be used by pharmacists in the treatment of patients with hypertension. DATA SOURCES: Published guidelines and abstracts identified through PubMed (May 1987 to April 2007) and Medline (January 1966 to April 2007) using the search terms hypertension, prehypertension, lifestyle modification, nutrition, physical activity, weight loss, weight control, behavior modification, smoking cessation, guidelines, and prevention, as well as the JNC 7 guidelines, NHLBI Obesity Guidelines, and Dietary Guidelines for Americans 2005. DATA SYNTHESIS: Lifestyle modification strategies are recommended in the JNC 7 guidelines for the treatment and prevention of hypertension and cardiovascular disease. The primary strategies discussed are proper nutrition through the Dietary Approaches to Stop Hypertension (DASH) eating plan and sodium restriction, weight reduction, increased physical activity, and moderation of alcohol consumption. Patients with hypertension have been shown to decrease their resting blood pressure considerably by adopting one of more of these strategies. CONCLUSION: Pharmacists are in an ideal setting to care for patients with hypertension by managing their medications and lifestyle behaviors. Doing so provides patients a higher level of clinical care from their pharmacist.

Therapeutic lifestyle strategies taught in U.S. Pharmacy Schools.

INTRODUCTION: Several organizations representing pharmacy and other health professions stress the importance of teaching public health topics as part of training future practitioners. The objective of our study was to assess the number of U.S. pharmacy schools that incorporate lifestyle modification topics into their curricula. METHODS: We developed an electronic survey on lifestyle modification topics and sent it to each of the 89 pharmacy schools in the United States. The survey defined lifestyle modification topics as topics that address nutrition, exercise, weight loss, smoking cessation, and alcohol use. RESULTS: Of 89 pharmacy schools contacted, 50 (56%) responded to the survey. Of the 50, four offer at least one required course in a lifestyle modification topic, seven offer at least one elective course, and one offers a required course that incorporates more than one lifestyle modification topic. Five required and nine elective courses were identified from the responses. Nutrition was the most commonly offered required course topic, followed by smoking cessation, exercise, weight loss, and alcohol use. CONCLUSION: Few pharmacy schools are addressing recommendations to promote public health education through formalized didactic courses. More courses on lifestyle modification topics should be offered to pharmacy students, who will be highly accessible to the public as pharmacists and will be able to offer education to enhance public health focused on the prevention of chronic diseases.

An improved nucleic acid parameter set for the GROMOS force field.

Over the past decades, the GROMOS force field for biomolecular simulation has primarily been developed for performing molecular dynamics (MD) simulations of polypeptides and, to a lesser extent, sugars. When applied to DNA, the 43A1 and 45A3 parameter sets of the years 1996 and 2001 produced rather flexible double-helical structures, in which the Watson-Crick hydrogen-bonding content was more limited than expected. To improve on the currently available parameter sets, the nucleotide backbone torsional-angle parameters and the charge distribution of the nucleotide bases are reconsidered based on quantum-chemical data. The new 45A4 parameter set resulting from this refinement appears to perform well in terms of reproducing solution NMR data and canonical hydrogen bonding. The deviation between simulated and experimental observables is now of the same order of magnitude as the uncertainty in the experimental values themselves.