Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12–cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.United States. National Institutes of Health (HG002668)United States. National Institutes of Health (CA109901

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12–cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.United States. National Institutes of Health (HG002668)United States. National Institutes of Health (CA109901

At the intersection of medicinal chemistry and chemical biology : development of chemical probes for target inhibition, degradation, and labeling

Chemical probes are important tools for target identification and validation studies that guide drug discovery and development. In this thesis, the development of chemical probes for therapeutically relevant protein targets is described. To achieve this, a wide variety of medicinal chemistry approaches were applied and include covalent and reversible protein inhibitors as well as targeted protein degradation strategies. In the main part (Part II), three chemical probe discovery projects, targeting the kinases PI5P4K and CDK12/13, and the isomerase Pin1, are presented. For all three protein targets, biotin/desthiobiotin-labeled probes of the covalent protein binders were developed and enabled the investigation of the target inhibitors’ scaffolds as potential leads for further drug development purposes or served to investigate target biology. In case of the PI5P4K inhibitors, two novel scaffolds were identified, starting from screening hits or kinase inhibitor off-targets. Medicinal chemistry optimization efforts led to improved potency and selectivity properties of initial hit compounds in vitro and in cellulo. In the Appendix (Part III), the development of unpublished probes is discussed, including a fragment-based probe targeting Pin1, a PAL-inspired probe to investigate the bacterial target MraY, and approaches to the development of PI5P4K- and Pin1-targeting protein degraders. While the MraY-targeting probe and the degrader molecules require further optimization, the presented work gives important insights into the development of these kinds of probes.Chemische Sonden stellen essentielle Werkzeuge für Zielproteinindentifikations- und Valdierungsstudien in der Entdeckung und Entwicklung von Arzneistoffen dar. In dieser Doktorarbeit wird die Entwicklung von chemischen Sonden für eine Reihe therapeutisch relevanter Zielproteine beschrieben. Hierfür wurden vielfältige medizinisch-chemische Ansätze, einschließlich kovalenter und reversibler Proteininhibitoren, sowie Strategien zum gezielten Proteinabbau, angewandt. Im Hauptteil (Part II) werden drei Sondenentwicklungsprojekte für die Inhibierung der Kinasen PI5P4K und CDK12/13, sowie der Isomerase Pin1 präsentiert. Für alle Zielproteine wurden Biotin/Desthiobiotin-markierte Sonden auf der Grundlage der entsprechenden Proteininhibitoren kreiert, welche zur Identifizierung der chemischen Verbindungen als potenzielle Leitstrukturen oder zur Untersuchung der Zielproteinbiologie dienten. Ausgehend von Screeninghits oder Off-targeteffekten von Kinaseninhibitoren konnten darüber hinaus zwei neuartige chemische Strukturserien als PI5P4K-Inhibitoren identifiziert werden. Medizinisch-chemische Optimierungen führten hier zu verbesserter Potenz und Selektivität der initialen Hits in vitro und in cellulo. Im Appendix (Part III) werden unpublizierte Sondenentwicklungsprojekte für eine Fragment-basierte Pin1-inhibierende Sonde, eine PAL-abgeleitete Sonde zur Untersuchung des bakteriellen Proteins MraY, sowie die Ansätze zur Entwicklung von PI5P4K- und Pin1- abbauenden Verbindungen diskutiert. Obwohl die MraY-inhibierende Sonde und die proteinabbauenden Verbindungen weitere Optimierung benötigen, gibt die hier dargelegte Arbeit wertvolle Einblicke in die Entwicklung dieser Sonden

Transboundary Monitoring of the Wolf Alpine Population over 21 Years and Seven Countries

Wolves have large spatial requirements and their expansion in Europe is occurring over national boundaries, hence the need to develop monitoring programs at the population level. Wolves in the Alps are defined as a functional population and management unit. The range of this wolf Alpine population now covers seven countries: Italy, France, Austria, Switzerland, Slovenia, Liechtenstein and Germany, making the development of a joint and coordinated monitoring program particularly challenging. In the framework of the Wolf Alpine Group (WAG), researchers developed uniform criteria for the assessment and interpretation of field data collected in the frame of different national monitoring programs. This standardization allowed for data comparability across borders and the joint evaluation of distribution and consistency at the population level. We documented the increase in the number of wolf reproductive units (packs and pairs) over 21 years, from 1 in 1993–1994 up to 243 units in 2020–2021, and examined the pattern of expansion over the Alps. This long-term and large-scale approach is a successful example of transboundary monitoring of a large carnivore population that, despite administrative fragmentation, provides robust indexes of population size and distribution that are of relevance for wolf conservation and management at the transnational Alpine scale

Junctional Adhesion Molecule-C expression specifies a {CD}138low/neg multiple myeloma cell population in mice and humans

Deregulation such as overexpression of adhesion molecules influences cancer progression and survival. Metastasis of malignant cells from their primary tumor site to distant organs is the most common reason for cancer-related deaths. Junctional adhesion molecule (JAM)-C, a member of the Ig-like JAM family, can homodimerize and aid cancer cell migration and metastasis. Here we show that this molecule is dynamically expressed on multiple myeloma (MM) cells in the marrow and co-localizes with blood vessels within the bone marrow of mice and humans. Additionally, JAM-C upregulation inversely correlates with the downregulation of the canonical plasma cell marker CD138 (syndecan-1), whose surface expression has recently been found to dynamically regulate a switch between MM growth in situ and MM dissemination. Moreover, targeting JAM-C in a syngeneic in vivo MM model ameliorates MM progression and improves outcome. Overall, our data demonstrate that JAM-C might serve not only as an additional novel diagnostic biomarker but also as a therapeutic target in MM disease

Transient regulatory T-cell targeting triggers immune control of multiple myeloma and prevents disease progression

Multiple myeloma remains a largely incurable disease of clonally expanding malignant plasma cells. The bone marrow microenvironment harbors treatment-resistant myeloma cells, which eventually lead to disease relapse in patients. In the bone marrow, CD4$^{+}$FoxP3$^{+}$ regulatory T cells (Tregs) are highly abundant amongst CD4$^{+}$ T cells providing an immune protective niche for different long-living cell populations, e.g., hematopoietic stem cells. Here, we addressed the functional role of Tregs in multiple myeloma dissemination to bone marrow compartments and disease progression. To investigate the immune regulation of multiple myeloma, we utilized syngeneic immunocompetent murine multiple myeloma models in two different genetic backgrounds. Analyzing the spatial immune architecture of multiple myeloma revealed that the bone marrow Tregs accumulated in the vicinity of malignant plasma cells and displayed an activated phenotype. In vivo Treg depletion prevented multiple myeloma dissemination in both models. Importantly, short-term in vivo depletion of Tregs in mice with established multiple myeloma evoked a potent CD8 T cell- and NK cell-mediated immune response resulting in complete and stable remission. Conclusively, this preclinical in-vivo study suggests that Tregs are an attractive target for the treatment of multiple myeloma

Transboundary Monitoring of the Wolf Alpine Population over 21 Years and Seven Countries

Wolves have large spatial requirements and their expansion in Europe is occurring over national boundaries, hence the need to develop monitoring programs at the population level. Wolves in the Alps are defined as a functional population and management unit. The range of this wolf Alpine population now covers seven countries: Italy, France, Austria, Switzerland, Slovenia, Liechtenstein and Germany, making the development of a joint and coordinated monitoring program particularly challenging. In the framework of the Wolf Alpine Group (WAG), researchers developed uniform criteria for the assessment and interpretation of field data collected in the frame of different national monitoring programs. This standardization allowed for data comparability across borders and the joint evaluation of distribution and consistency at the population level. We documented the increase in the number of wolf reproductive units (packs and pairs) over 21 years, from 1 in 1993-1994 up to 243 units in 2020-2021, and examined the pattern of expansion over the Alps. This long-term and large-scale approach is a successful example of transboundary monitoring of a large carnivore population that, despite administrative fragmentation, provides robust indexes of population size and distribution that are of relevance for wolf conservation and management at the transnational Alpine scale

Human Cyclin-dependent kinase 12 (CDK12), kinase domain; A Target Enabling Package

<p>Cyclin-dependent kinase 12 (CDK12) phosphorylates RNA Pol II C-terminal domain (CTD) to promote transcriptional elongation of large DNA damage response genes. CDK12 is frequently mutated or amplified in cancer and its loss sensitises cells to DNA damage. Here we present 3 crystal structures of the human CDK12/CycK complex including apo, AMP-PNP and covalent inhibitor complexes. Kinase assays compare domain truncations and report the Km values for substrate. THZ531 is presented as a potent and selective inhibitor of CDK12 with nanomolar activity in leukemic cell lines.</p