In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Resistance to chemotherapy and molecularly targeted therapies is a major problem facing current cancer research. The mechanisms of resistance to 'classical' cytotoxic chemotherapeutics and to therapies that are designed to be selective for specific molecular targets share many features, such as alterations in the drug target, activation of prosurvival pathways and ineffective induction of cell death. With the increasing arsenal of anticancer agents, improving preclinical models and the advent of powerful high-throughput screening techniques, there are now unprecedented opportunities to understand and overcome drug resistance through the clinical assessment of rational therapeutic drug combinations and the use of predictive biomarkers to enable patient stratification.

Cancer drug resistance: an evolving paradigm

Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.

Mitochondrial Membrane Permeabilization in Cell Death

BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-Myc

The BCL-2 protein family determines the commitment of cells to apoptosis, an ancient cell suicide programme that is essential for development, tissue homeostasis and immunity. Too little apoptosis can promote cancer and autoimmune diseases; too much apoptosis can augment ischaemic conditions and drive neurodegeneration. We discuss the biochemical, structural and genetic studies that have clarified how the interplay between members of the BCL-2 family on mitochondria sets the apoptotic threshold. These mechanistic insights into the functions of the BCL-2 family are illuminating the physiological control of apoptosis, the pathological consequences of its dysregulation and the promising search for novel cancer therapies that target the BCL-2 family.

Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy

FIELD: pharmacology.SUBSTANCE: invention relates to organic chemistry, namely a heterocyclic compound of formula(I) or to a therapeutically acceptable salt thereof, where X is benzo[d]thiazolyl, thiazolo[5,4-b]pyridinyl, thiazolo[4,5-c]pyridinyl, imidazo[1,2-a]pyridinyl, thiazolo[5,4-c]pyridinyl, thiazolo[4,5-b]pyridinyl, imidazo[1,2-a]pyrazinyl or imidazo[ 1,2-b]pyridazinyl; Ymeans pyrrolyl, pyrazolyl, triazolyl or pyridinyl; where Y1 is optionally substituted with 1 or 2 substituents independently selected from R, CN and Cl; Lis selected from (CRR), (CRR)-O-(CRR), (CRR)-S-(CRR), (CRR)-S(O)-(CRR)and (CRR)-NR-(CRR); Ymeans mono- or tricyclic C-C-cycloalkyl, spiro[2.5]octyl, spiro[3.5]nonyl, oxatricyclo[3.3.1.1]decyl or azabicyclo[3.2.1]octyl; where Yis optionally substituted with 1, 2 or 3 substituents independently selected from R, OR, SOR, CO(O)R, OH and Br; Zis selected from, Rand Rare absent; Ris deuterium or C-C-alkyl; Ris C-C-alkyl; Ris selected from hydrogen and C-C-alkyl; each Rand Rare hydrogen; Ris selected from the group consisting of C-C-alkyl, morpholinyl and dioxydothiomorpholinyl; where R, meaning C-C-alkyl, is optionally substituted with a substituent selected from R, OR, SORand NHR; Ris selected from C-C-alkyl, morpholinyl, cyclopropyl and C-haloalkyl; RIs selected from C-C-alkyl, phenyl, morpholinyl and tetrahydropyranyl; where R, meaning C-C-alkyl, is optionally substituted with 1 substituent selected from OCH, OCHCHOCHand OCHCHNHCH; q is 1 or 2; s, m and p is 0; r is 0 or 1; N is 0, 1, or 2. The invention also relates to specific compounds, pharmaceutical compositions based on these compounds and a method for treatment of these diseases.EFFECT: new heterocyclic compounds are obtained, that are useful for treatment of some cancers.16 cl, 3 tbl, 86 ex

8-carbamoyl-2-(2,3-di-substituted pyrid-6-yl)-1,2,3,4-tetrahydroisoquinoline derivatives as apoptosis-inducing agents for cancer treatment

A Facile One-Carbon Homologation of Aryl Aldehydes to Amides.

L'invention concerne des composes qui inhibent l'activite de proteines Bcl-2 anti-apoptotiques, des compositions contenant ces composes et des methodes de traitement de maladies dans lesquelles la proteine Bcl-2 anti-apoptotique est exprimee.

Agents induisant l'apoptose pour le traitement du cancer et de maladies immunes et auto-immunes

La presente invention concerne des inhibiteurs de Bcl-xL ayant une faible permeabilite cellulaire, des conjugues anticorps-medicament (ADC) comprenant les inhibiteurs, des synthons utiles pour la synthese des ADC, des compositions comportant les inhibiteurs ou les ADC, et divers procedes d'utilisation des inhibiteurs et des ADC.

Composés inhibiteurs de bcl xl ayant une faible perméabilité cellulaire et conjugués anticorps-médicament comprenant ceux-ci

La presente invention concerne des inhibiteurs de Bcl-xL a petites molecules et des conjugues anticorps-medicament (ADC) comprenant les inhibiteurs de Bcl-xL a petites molecules. Les inhibiteurs de Bcl-xL et les ADC de l'invention sont utiles, entre autres, pour l'inhibition de proteines anti-apoptotiques Bcl-xL en tant qu'approche therapeutique pour le traitement de maladies qui impliquent une voie de l'apoptose deregulee.

Kunzer Aaron R

Papers

8-carbamoyl-2-(2,3-di-substituted pyrid-6-yl)-1,2,3,4-tetrahydroisoquinoline derivatives as apoptosis-inducing agents for cancer treatment

A Facile One-Carbon Homologation of Aryl Aldehydes to Amides.

Agents induisant l'apoptose pour le traitement du cancer et de maladies immunes et auto-immunes

Composés inhibiteurs de bcl xl ayant une faible perméabilité cellulaire et conjugués anticorps-médicament comprenant ceux-ci

Composés inhibiteurs de bcl-xl et conjugués anticorps-médicament comprenant ceux-ci