There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/Spy-Catcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.
Background and Purpose The TRPC1, TRPC4, and TRPC5 proteins form homotetrameric or heterotetrameric, calcium‐permeable cation channels that are involved in various disease states. Recent research has yielded specific and potent xanthine‐based TRPC1/4/5 inhibitors. Here, we investigated the possibility of xanthine‐based activators of these channels. Experimental Approach An analogue of the TRPC1/4/5 inhibitor Pico145, AM237, was synthesized and its activity was investigated using HEK cells overexpressing TRPC4, TRPC5, TRPC4–C1, TRPC5–C1, TRPC1:C4 or TRPC1:C5 channels, and in A498 cells expressing native TRPC1:C4 channels. TRPC1/4/5 channel activities were assayed by measuring intracellular concentration of Ca2+ ([Ca2+]i) and by patch‐clamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4, or TRPM2 channels. Key Results AM237 potently activated TRPC5:C5 channels (EC50 15–20 nM in [Ca2+]i assay) and potentiated their activation by sphingosine‐1‐phosphate but suppressed activation evoked by (−)‐englerin A (EA). In patch‐clamp studies, AM237 activated TRPC5:C5 channels, with greater effect at positive voltages, but with lower efficacy than EA. Pico145 competitively inhibited AM237‐induced TRPC5:C5 activation. AM237 did not activate TRPC4:C4, TRPC4–C1, TRPC5–C1, TRPC1:C5, and TRPC1:C4 channels, or native TRPC1:C4 channels in A498 cells, but potently inhibited EA‐dependent activation of these channels with IC50 values ranging from 0.9 to 7 nM. AM237 (300 nM) did not activate or inhibit TRPC3, TRPC6, TRPV4, or TRPM2 channels. Conclusions and Implications This study suggests the possibility for selective activation of TRPC5 channels by xanthine derivatives and supports the general principle that xanthine‐based compounds can activate, potentiate, or inhibit these channels depending on subunit composition.
Pharmacological inhibition of uncontrolled cell growth with small-molecule inhibitors is a potential strategy for treating glioblastoma multiforme (GBM), the most malignant primary brain cancer. We showed that the synthetic small-molecule KHS101 promoted tumor cell death in diverse GBM cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM.
Ein gemeinsames Erkennungsmotiv aus einer negativ geladenen Gruppe (rot) sechs bis sieben Bindungen entfernt von der (Thio)esterfunktion (grün) und einer positiv geladenen Schwanzgruppe (blau) zehn bis zwölf Bindungen entfernt wurde in zwei nativen Substraten der Acyl‐Protein‐Thioesterase 1 (APT1) identifiziert (siehe Bild). Diese Ähnlichkeit führte zum Design potenter Inhibitoren des Ras‐depalmitoylierenden Enzyms APT1.
Welche Substanzklassen eignen sich am besten als Werkzeuge für chemisch‐biologische Forschung und als Inspiration für medizinalchemische Projekte? Der chemische Strukturraum ist riesig und kann durch Synthese von Verbindungen nicht vollständig exploriert werden. Daher werden Methoden benötigt, mit deren Hilfe die biologisch relevanten Anteile des chemischen Strukturraums identifiziert und kartiert werden können. Die mit diesen Methoden erzeugten Hypothesen inspirieren dann Syntheseprogramme, um die biologisch relevanten Teile des Strukturraumes mit realen Verbindungen zu füllen. Die Biologie‐orientierte Synthese baut darauf auf, dass die Evolution von Proteinen und Naturstoffen sich auf bestimmte Strukturklassen beschränkt. Sie nutzt eine hierarchische Klassifikation bioaktiver Substanzen, die auf Substrukturbeziehungen und der Art der biologischen Aktivität beruht. Mit dieser Methode werden Gerüststrukturen biologisch aktiver Substanzklassen ausgewählt und als Startpunkte für die Synthese von Substanzkollektionen mit fokussierter Diversität genutzt. Scaffold Hunter, ein intuitiv zugängliches und hochgradig interaktives Computerprogramm, ermöglicht die Navigation im chemischen Strukturraum. In Bibliotheken niedermolekularer Substanzen, die nach dem BIOS‐Konzept synthetisiert wurden, ist biologische Aktivität oft angereichert. Sie ermöglichen die Untersuchung komplexer biologischer Phänomene durch direkte Perturbation und können darüber hinaus auch als Inspiration in der Medikamentenentwicklung dienen.
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