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Modulating chemoselectivity in a Fe(II)/ alpha-ketoglutarate dependent oxygenase for the oxidative modification of a non-proteinogenic amino acid
No full textModification of aliphatic C-H bonds in a regio- and stereoselective manner can pose a formidable challenge as these are least reactive in organic chemistry. In this context, the use of non-heme iron and α-ketoglutarate-dependent dioxygenases (αKGDs) represents an interesting complementary tool as this enzyme family can catalyze a broad set of synthetically valuable reactions including hydroxylations, oxidations and desaturations. The consensus reaction mechanism of this enzyme family proceeds via the formation of a Fe(IV)-oxo complex capable of hydrogen atom transfer (HAT) from a sp3- hybridized substrate carbon center. The resulting substrate radical and Fe(III)-OH cofactor is considered to be the branch point toward the possible reaction outcomes which are determined by the enzyme’s active site architecture. To date, the modulation of the reaction fate in Fe/ α-ketoglutarate-dependent oxygenases via enzyme engineering has been mainly elusive. In this study, we therefore targeted to engineer the L-proline cis-4-hydroxylase SmP4H from Sinorhizobium meliloti for selective oxidative modification of the non-proteinogenic amino acid L-homo-phenylalanine (L-hPhe) to produce pharmacological relevant small molecule intermediates. Using structure-guided directed evolution, we improved the turnover number and apparent kcat of the hydroxylation reaction yielding the desired -hydroxylation product by approximately 10-fold and 20-fold, respectively. Notably, the introduction of only one new catalytic entity into the active site (W40Y), allowed us to re-program the natural hydroxylase to predominantly act as a desaturase, presumably through tyrosine’s capability to serve as a catalytic base in the reaction mechanism
Role of Tumor-Infiltrating B Cells in Clinical Outcome of Patients with Melanoma Treated With Dabrafenib Plus Trametinib
No full textPURPOSE: Although patients with unresectable or metastatic melanoma can experience long-term survival with BRAF- and MEK-targeted agents or immune checkpoint inhibitors over 5 years, resistance develops in most patients. There is a distinct lack of pretherapeutic biomarkers to identify which patients are likely to benefit from each therapy type. Most research has focused on the predictive role of T cells in antitumor responses as opposed to B cells. EXPERIMENTAL DESIGN: We conducted prespecified exploratory biomarker analysis using gene expression profiling and digital pathology in 146 patients with previously untreated BRAF V600-mutant metastatic melanoma from the randomized, phase III COMBI-v trial and treated with dabrafenib plus trametinib who had available tumor specimens from screening. RESULTS: Baseline cell cycle gene expression signature was associated with progression-free survival (P = 0.007). Patients with high T-cell/low B-cell gene signatures had improved median overall survival (not reached [95% confidence interval (CI), 33.8 months-not reached]) compared with patients with high T-cell/high B-cell signatures (19.1 months [95% CI, 13.4-38.6 months]). Patients with high B-cell signatures had high B-cell infiltration into the tumor compartment, corresponding with decreased mitogen-activated protein kinase activity and increased expression of immunosuppressive markers. CONCLUSIONS: B cells may serve as a potential biomarker to predict clinical outcome in patients with advanced melanoma treated with dabrafenib plus trametinib. As separate studies have shown an opposite effect for B-cell levels and response to immunotherapy, B cells may serve as a potential biomarker to facilitate treatment selection. Further validation in a larger patient cohort is needed
Structure-based design of selective LONP1 inhibitors for probing in vitro biology
No full textLONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology
Passive influx and ion trapping are more relevant to the cellular accumulation of highly permeable low molecular weight acidic drugs than is Organic Anion Transporter 2 (OAT2)
No full textRecently published work suggests that highly permeable low molecular weight (LMW) acidic drugs are transported by Organic Anion Transporter 2 (OAT2). However, an asymmetric distribution of ionizable drugs in subcellular organelles where pH gradients are significant may occur in the presence of an inhibitor relative to its absence (e.g. lysosomal trapping). In the present study, OAT2-mediated transport of highly permeable LMW anions could not be demonstrated using OAT2 transfected cells, despite robust transport of the OAT2 substrate penciclovir. Moreover, a rifamycin SV (RifSV) dependent reduction in the accumulation of highly permeable LMW anions previously observed in hepatocytes could be qualitatively reproduced using HepG2 cells and also in MDCK cells which lack expression of OAT2. Neither HepG2 nor MDCK cells demonstrated meaningful penciclovir transport, nor was the cellular accumulation of the highly permeable LMW anions sensitive to competitive inhibition by the neutral OAT2 substrate penciclovir. Both cell lines however demonstrated sensitivity to the mitochondrial uncoupler p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) in a manner similar to RifSV. Furthermore, the transepithelial MDCK permeability of the highly permeable LMW anions was measured in the absence and presence of RifSV and FCCP at concentrations that reduced the cellular accumulation of anions. Neither inhibitor, nor the OAT2 inhibitor ketoprofen, reduced the transepithelial flux of the anions as would be anticipated for transported substrate inhibition. The findings presented here are aligned with cellular accumulation of highly permeable LMW anions being significantly determined by ion trapping sensitive to mitochondrial uncoupling rather than the result of OAT2-mediated transport. Significance Statement The manuscript illustrates that passive influx and ion trapping are more relevant to the cellular accumulation of highly permeable low molecular weight acidic drugs than is the previously proposed mechanism of OAT2-mediated transport. The outcome illustrated here highlights a rare, and perhaps previously not reported, observation of anionic drug trapping in a compartment sensitive to mitochondrial uncoupling (e.g. the mitochondrial matrix) that may be confused for transporter-mediated uptake
Antitarget selectivity and tolerability of novel pyrrolo[2,3-d]pyrimidine RET inhibitors
No full textThe selective inhibition of RET kinase as a treatment for relevant cancer types including lung adenocarcinoma has garnered considerable interest in recent years and prompted a variety of efforts toward the discovery of small-molecule therapeutics. Hits uncovered via the analysis of archival kinase data ultimately led to the identification of a promising pyrrolo[2,3-d]pyrimidine scaffold. The optimization of this pyrrolo[2,3-d]pyrimidine core resulted in compound 1, which demonstrated potent in vitro RET kinase inhibition and robust in vivo efficacy in RET-driven tumor xenografts upon multiday dosing in mice. The administration of 1 was well-tolerated at established efficacious doses (10 and 30 mg/kg, po, qd), and plasma exposure levels indicated a minimal risk of KDR or hERG inhibition in vivo, as evaluated by Miles assay and free plasma concentrations, respectively
FAIRification at Novartis, published as one section in Why FAIR?: A course module for the IMI FAIRplus Fellowship training program
No full textFAIRification at Novartis, published as one section in Why FAIR?: A course module for the IMI FAIRplus Fellowship training program
The FAIRplus fellowship training program is one output of the IMI FAIRplus project. FAIRification at Novartis is an overview of selected Novartis Data FAIRification initiatives
The Full course module is available at the following link:https://ilias.fraunhofer.de/ilias.php?baseClass=ilSAHSPresentationGUI&ref_id=1715
Matched Targeted Therapy for Pediatric Patients with Relapsed, Refractory, or High-Risk Leukemias: A Report from the LEAP Consortium
No full textDespite a remarkable increase in the genomic profiling of cancer, integration of genomic discoveries into clinical care has lagged behind. We report the feasibility of rapid identification of targetable mutations in 153 pediatric patients with relapsed/refractory or high-risk leukemias enrolled on a prospective clinical trial conducted by the LEAP Consortium. Eighteen percent of patients had a high confidence Tier 1 or 2 recommendation. We describe clinical responses in the 14% of patients with relapsed/refractory leukemia who received the matched targeted therapy. Further, in order to inform future targeted therapy for patients, we validated variants of uncertain significance, performed ex vivo drug-sensitivity testing in patient leukemia samples, and identified new combinations of targeted therapies in cell lines and patient-derived xenograft models. These data and our collaborative approach should inform the design of future precision medicine trials
BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells
No full textThe transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1; Fos–Jun) cooperate to promote
the effector functions of T cells, but NFAT in the absence of AP-1 imposes a negative feedback program of T cell hyporesponsiveness (exhaustion). Here, we show that basic leucine zipper ATF-like transcription factor (BATF) and interferon regulatory
factor 4 (IRF4) cooperate to counter T cell exhaustion in mouse tumor models. Overexpression of BATF in CD8+ T cells expressing a chimeric antigen receptor (CAR) promoted the survival and expansion of tumor-infiltrating CAR T cells, increased the
production of effector cytokines, decreased the expression of inhibitory receptors and the exhaustion-associated transcription
factor TOX and supported the generation of long-lived memory T cells that controlled tumor recurrence. These responses were
dependent on BATF–IRF interaction, since cells expressing a BATF variant unable to interact with IRF4 did not survive in tumors
and did not effectively delay tumor growth. BATF may improve the antitumor responses of CAR T cells by skewing their phenotypes and transcriptional profiles away from exhaustion and towards increased effector function
Best practices for repurposing studies.
No full textll I can say is that on this earth there are pestilences
and there are victims– and as far as possible one must
refuse to be on the side of the pestilence.
—Albert Camus, La Peste [1].
As humanity is challenged with new pandemics and diseases,
there is a desire to find treatments in a much faster
time-frame than traditional discovery efforts can attain. One
strategy has been the repurposing of compounds that have
demonstrated efficacy and/or safety in human trials (see the
articles in the special issue of Drug Discovery Today [2]) or
through common usage. The goal is to identify compounds
that may proceed directly to human efficacy studies. If a
compound requires optimisation, then all its advantages are
gone, and there is little to choose between it and any other
hit structure, in terms of time to the clinic, the key metric.
During the current Covid-19 pandemic, many papers have
been written with the aim of identifying possible candidates
for the repurposing strategy, but which unfortunately have
been lacking in the proper controls or understanding to be
accepted in this journal. This brief editorial tries to lay out
some of the issues that authors should address in their studies.
We do not hold a position for or against repurposing,
but we must try to maintain certain scientific standards on
behalf of our readership. We also have a duty not to mislead,
resulting in studies that cost much human effort and divert
resources from more promising areas, but to inform with
genuine data
Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum.
No full textOur current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs