Jeffrey D. Winkler

Bio: Jeffrey D. Winkler is an academic researcher from University of Pennsylvania. The author has contributed to research in topics: Ring (chemistry) & Autophagy. The author has an hindex of 44, co-authored 204 publications receiving 6573 citations. Previous affiliations of Jeffrey D. Winkler include Merck & Co. & Florida State University College of Arts and Sciences.

Targeting ER stress–induced autophagy overcomes BRAF inhibitor resistance in melanoma

Abstract: Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAFV600E melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAFV600E melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.

Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency

Abstract: Autophagy is a lysosome-dependent degradative process that protects cancer cells from multiple stresses. In preclinical models, autophagy inhibition with chloroquine (CQ) derivatives augments the efficacy of many anticancer therapies, but CQ has limited activity as a single agent. Clinical trials are underway combining anticancer agents with hydroxychloroquine (HCQ), but concentrations of HCQ required to inhibit autophagy are not consistently achievable in the clinic. We report the synthesis and characterization of bisaminoquinoline autophagy inhibitors that potently inhibit autophagy and impair tumor growth in vivo. The structural motifs that are necessary for improved autophagy inhibition compared with CQ include the presence of two aminoquinoline rings and a triamine linker and C-7 chlorine. The lead compound, Lys01, is a 10-fold more potent autophagy inhibitor than HCQ. Compared with HCQ, Lys05, a water-soluble salt of Lys01, more potently accumulates within and deacidifies the lysosome, resulting in impaired autophagy and tumor growth. At the highest dose administered, some mice develop Paneth cell dysfunction that resembles the intestinal phenotype of mice and humans with genetic defects in the autophagy gene ATG16L1, providing in vivo evidence that Lys05 targets autophagy. Unlike HCQ, significant single-agent antitumor activity is observed without toxicity in mice treated with lower doses of Lys05, establishing the therapeutic potential of this compound in cancer.

De novo design and in vivo activity of conformationally restrained antimicrobial arylamide foldamers

Abstract: The emergence of drug-resistant bacteria has compromised the use of many conventional antibiotics, leading to heightened interest in a variety of antimicrobial peptides. Although these peptides have attractive potential as antibiotics, their size, stability, tissue distribution, and toxicity have hampered attempts to harness these capabilities. To address such issues, we have developed small (molecular mass <1,000 Da) arylamide foldamers that mimic antimicrobial peptides. Hydrogen-bonded restraints in the arylamide template rigidify the conformation via hydrogen bond formation and increase activity toward Staphylococcus aureus and Escherichia coli. The designed foldamers are highly active against S. aureus in an animal model. These results demonstrate the application of foldamer templates as therapeutics.

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

Abstract: 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.

Patterns of Somatic Mutation in Human Cancer Genomes

Abstract: AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore PL02-05 All cancers are due to abnormalities in DNA. The availability of the human genome sequence has led to the proposal that resequencing of cancer genomes will reveal the full complement of somatic mutations and hence all the cancer genes. To explore the nature of the information that will be derived from cancer genome sequencing we have sequenced the coding exons of the family of 518 protein kinases, ~1.3Mb DNA per cancer sample, in 210 cancers of diverse histological types. Despite the screen being directed toward the coding regions of a gene family that has previously been strongly implicated in oncogenesis, the results indicate that the majority of somatic mutations detected are “passengers”. There is considerable variation in the number and pattern of these mutations between individual cancers, indicating substantial diversity of processes of molecular evolution between cancers. The imprints of exogenous mutagenic exposures, mutagenic treatment regimes and DNA repair defects can all be seen in the distinctive mutational signatures of individual cancers. This systematic mutation screen and others have previously yielded a number of cancer genes that are frequently mutated in one or more cancer types and which are now anticancer drug targets (for example BRAF , PIK3CA , and EGFR ). However, detailed analyses of the data from our screen additionally suggest that there exist a large number of additional “driver” mutations which are distributed across a substantial number of genes. It therefore appears that cells may be able to utilise mutations in a large repertoire of potential cancer genes to acquire the neoplastic phenotype. However, many of these genes are employed only infrequently. These findings may have implications for future anticancer drug development.

C-H bond functionalization: emerging synthetic tools for natural products and pharmaceuticals

Abstract: The direct functionalization of C-H bonds in organic compounds has recently emerged as a powerful and ideal method for the formation of carbon-carbon and carbon-heteroatom bonds. This Review provides an overview of C-H bond functionalization strategies for the rapid synthesis of biologically active compounds such as natural products and pharmaceutical targets.

Palladium-catalyzed cross-coupling reactions in total synthesis.

Abstract: In studying the evolution of organic chemistry and grasping its essence, one comes quickly to the conclusion that no other type of reaction plays as large a role in shaping this domain of science than carbon-carbon bond-forming reactions. The Grignard, Diels-Alder, and Wittig reactions are but three prominent examples of such processes, and are among those which have undeniably exercised decisive roles in the last century in the emergence of chemical synthesis as we know it today. In the last quarter of the 20th century, a new family of carbon-carbon bond-forming reactions based on transition-metal catalysts evolved as powerful tools in synthesis. Among them, the palladium-catalyzed cross-coupling reactions are the most prominent. In this Review, highlights of a number of selected syntheses are discussed. The examples chosen demonstrate the enormous power of these processes in the art of total synthesis and underscore their future potential in chemical synthesis.

Sildenafil citrate therapy for pulmonary arterial hypertension.

Abstract: background Sildenafil inhibits phosphodiesterase type 5, an enzyme that metabolizes cyclic guanosine monophosphate, thereby enhancing the cyclic guanosine monophosphate– mediated relaxation and growth inhibition of vascular smooth-muscle cells, including those in the lung. methods In this double-blind, placebo-controlled study, we randomly assigned 278 patients with symptomatic pulmonary arterial hypertension (either idiopathic or associated with connective-tissue disease or with repaired congenital systemic-to-pulmonary shunts) to placebo or sildenafil (20, 40, or 80 mg) orally three times daily for 12 weeks. The primary end point was the change from baseline to week 12 in the distance walked in six minutes. The change in mean pulmonary-artery pressure and World Health Organization (WHO) functional class and the incidence of clinical worsening were also assessed, but the study was not powered to assess mortality. Patients completing the 12-week randomized study could enter a long-term extension study. results The distance walked in six minutes increased from baseline in all sildenafil groups; the mean placebo-corrected treatment effects were 45 m (+13.0 percent), 46 m (+13.3 percent), and 50 m (+14.7 percent) for 20, 40, and 80 mg of sildenafil, respectively (P<0.001 for all comparisons). All sildenafil doses reduced the mean pulmonary-artery pressure (P = 0.04, P = 0.01, and P<0.001, respectively), improved the WHO functional class (P = 0.003, P<0.001, and P<0.001, respectively), and were associated with side effects such as flushing, dyspepsia, and diarrhea. The incidence of clinical worsening did not differ significantly between the patients treated with sildenafil and those treated with placebo. Among the 222 patients completing one year of treatment with sildenafil monotherapy, the improvement from baseline at one year in the distance walked in six minutes was 51 m. conclusions Sildenafil improves exercise capacity, WHO functional class, and hemodynamics in patients with symptomatic pulmonary arterial hypertension.