Tetrazole-containing STAT5 Inhibitors Derived from Furazan-based Phosphate Mimetics

Signal transducers and activators of transcription (STAT) are a family of proteins that regulate gene transcription. Seven members (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6) have been identified in human STAT family since two decades ago. As a member of the STAT family, STAT5 plays a central role in transmitting signals from transmembrane receptors such as cytokine receptors and growth factor receptors to the nucleus. It has been found that STAT5 is constitutively phosphorylated at a conserved tyrosine residue located at the end of SH2 domain by various activated tyrosine kinases in a broad range of human cancer cells. Inhibiting STAT5 has been considered as a promising approach for cancer therapy. However, STATs have been described as difficult pharmacological targets and small-molecule inhibitors of STAT5 are poorly investigated. The subject of this work is the investigation of small-molecule inhibitors targeting STAT5b-SH2. The tetrazole-containing active compounds discovered in this work are derived from the furazan-based phosphate mimetics. In the first section of this work, a structurally stable scaffold was designed. The aim is to improve the binding affinity of the existing inhibitors and to avoid the instability. For this purpose, four different methods were tried to synthesize the new structures. The main problem, low reactivity of the amino group in fragment 1 in the formation of a linkage between the furazan ring and the 5-position in the tetrazole ring, was solved in the N-cyanomethyl amine synthetic method. The obtained compounds showed expected stability under acidic conditions. However, these compounds were surprisingly inactive in a FP binding assay. In the second section of this work, a series of 1-aminomethyl tetrazoles based on the structures with different substituents at the 5-position of the tetrazole ring was synthesized via Mannich ligation reaction. Most of them displayed binding affinity to STAT5b-SH2 in low micromolar range in the FP-based competitive binding assay, in which compound 24 was the most active one with a Ki value of 1.8 µM. To investigate how different substituents at the 5-position of the tetrazole ring influence the stability of the scaffold, a systematic stability analysis was carried out on LC-MS and compounds 19, 22, 24, and 25 were found to possess better stability in contrast with the lead compounds 2 and 3. Furthermore, the binding of compound 24 to STAT5b-SH2 and the pivotal role of Asn642 were proven in the binding assays. Finally, the inhibitory activity of compounds 3 and 24 on leukemic cell proliferation was determined by the Alamar Blue assay.STAT (signal transducers and activators of transcription) sind eine Familie von Proteinen, die die Gentranskription regulieren. Sieben Mitglieder (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6) wurden vor zwei Jahrzehnten in der menschlichen STAT-Familie identifiziert. Als Mitglied der STAT-Familie spielt STAT5 eine zentrale Rolle bei der Übertragung von Signalen von Transmembranrezeptoren wie Zytokinrezeptoren und Wachstumsfaktorrezeptoren zum Zellkern. Es wurde gefunden, dass STAT5 an einem konservierten Tyrosinrest, der sich am Ende der SH2-Domäne befindet, durch verschiedene aktivierte Tyrosinkinasen in einem breiten Bereich menschlicher Krebszellen konstitutiv phosphoryliert wird. Die Hemmung von STAT5 gilt als vielversprechender Ansatz für die Krebstherapie. STAT-Proteine wurden jedoch als schwierige pharmakologische Ziele beschrieben, und niedermolekulare Inhibitoren von STAT5 sind nur unzureichend untersucht. Gegenstand dieser Arbeit ist die Untersuchung von niedermolekularen Inhibitoren, die auf STAT5b-SH2 abzielen. Die in dieser Arbeit entdeckten Tetrazol-haltigen Verbindungen leiten sich von den Furazan-basierten Phosphat-Mimetika ab. Im ersten Teil dieser Arbeit wurde ein strukturelles stabiles Gerüst entworfen. Ziel ist es, die Bindungsaffinität der vorhandenen Inhibitoren zu verbessern und die Instabilität zu vermeiden. Zu diesem Zweck wurden vier verschiedene Methoden zur Synthese der neuen Strukturen ausprobiert. Das Hauptproblem, die geringe Reaktivität der Aminogruppe in Fragment 1 bei der Bildung einer Bindung zwischen dem Furazanring und der 5-Position im Tetrazolring, wurde mit der N-Cyanomethylamin-Synthesemethode gelöst. Die erhaltenen Verbindungen zeigten erwartete Stabilität unter sauren Bedingungen. Diese Verbindungen waren jedoch in einem FP-Bindungsassay überraschend inaktiv. Im zweiten Teil dieser Arbeit wurde eine Reihe von 1-Aminomethyltetrazolen basierend auf den Strukturen mit unterschiedlichen Substituenten an der 5-Position des Tetrazolrings durch Mannich-Ligation synthetisiert. Die meisten von ihnen zeigten im FP-basierten kompetitiven Bindungstest, in dem Verbindung 24 die aktivste Verbindung mit einem Ki-Wert von 1,8 µM war, eine Bindungsaffinität zu STAT5b-SH2 im niedrigen mikromolaren Bereich. Um zu untersuchen, wie unterschiedliche Substituenten an der 5-Position des Tetrazolrings die Stabilität des Gerüsts beeinflussen, wurde eine systematische Stabilitätsanalyse an LC-MS durchgeführt und festgestellt, dass die Verbindungen 19, 22, 24 und 25 im Gegensatz dazu eine bessere Stabilität aufweisen mit den Bleiverbindungen 2 und 3. Weiterhin wurden die Bindung von Verbindung 24 an STAT5b-SH2 und die Schlüsselrolle von Asn642 in den Bindungsassays nachgewiesen. Schließlich wurde die Hemmwirkung der Verbindungen 3 und 24 auf die Leukämiezellproliferation durch den Alamar Blue-Assay bestimmt

Multistage signal-interactive nanoparticles improve tumor targeting through efficient nanoparticle-cell communications

Communication between biological components is critical for homeostasis maintenance among the convergence of complicated bio-signals. For therapeutic nanoparticles (NPs), the general lack of effective communication mechanisms with the external cellular environment causes loss of homeostasis, resulting in deprived autonomy, severe macrophage-mediated clearance, and limited tumor accumulation. Here, we develop a multistage signal-interactive system on porous silicon particles through integrating the Self-peptide and Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide into a hierarchical chimeric signaling interface with “don’t eat me” and “eat me” signals. This biochemical transceiver can act as both the signal receiver for amantadine to achieve NP transformation and signal conversion as well as the signal source to present different signals sequentially by reversible self-mimicking. Compared with the non-interactive controls, these signal-interactive NPs loaded with AS1411 and tanespimycin (17-AAG) as anticancer drugs improve tumor targeting 2.8-fold and tumor suppression 6.5-fold and showed only 51% accumulation in the liver with restricted hepatic injury.Peer reviewe

Targeting STAT3 and STAT5 in Cancer

Every minute, 34 new patients are diagnosed with cancer globally. Although over the past 50 years treatments have improved and survival rates have increased dramatically for several types of cancers, many remain incurable. Several aggressive types of blood and solid cancers form when mutations occur in a critical cellular signaling pathway, the JAK-STAT pathway; (Janus Kinase-Signal Transducer and Activator of Transcription). Currently, there are no clinically available drugs that target the oncogenic STAT3/5 proteins in particular or their Gain of Function hyperactive mutant products. Here, we summarize targeting approaches on STAT3/5, as the field moves towards clinical applications as well as we illuminate on upstream or downstream JAK-STAT pathway interference with kinase inhibitors, heat shock protein blockers or changing nuclear import/export processes. We cover the design paradigms and medicinal chemistry approaches to illuminate progress and challenges in understanding the pleiotropic role of STAT3 and STAT5 in oncogenesis, the microenvironment, the immune system in particular, all culminating in a complex interplay towards cancer progression

Multistage signal-interactive nanoparticles improve tumor targeting through efficient nanoparticle-cell communications

Communication between biological components is critical for homeostasis maintenance among the convergence of complicated bio-signals. For therapeutic nanoparticles (NPs), the general lack of effective communication mechanisms with the external cellular environment causes loss of homeostasis, resulting in deprived autonomy, severe macrophage-mediated clearance, and limited tumor accumulation. Here, we develop a multistage signal-interactive system on porous silicon particles through integrating the Self-peptide and Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide into a hierarchical chimeric signaling interface with “don’t eat me” and “eat me” signals. This biochemical transceiver can act as both the signal receiver for amantadine to achieve NP transformation and signal conversion as well as the signal source to present different signals sequentially by reversible self-mimicking. Compared with the non-interactive controls, these signal-interactive NPs loaded with AS1411 and tanespimycin (17-AAG) as anticancer drugs improve tumor targeting 2.8-fold and tumor suppression 6.5-fold and showed only 51% accumulation in the liver with restricted hepatic injury.</p

Multistage signal-interactive nanoparticles improve tumor targeting through efficient nanoparticle-cell communications

Supramolecular & Biomaterials Chemistr

Chromatin alterations during transformation of B cells by a constitutively active mutant of STAT5

The transcription factor signal transducer and activator of transcription 5 (STAT5) is activated conditionally and transiently by external stimuli. Thereupon, STAT5 modulates the transcription of its target genes, promoting cell survival and growth. Constitutive STAT5 activity has been shown to be oncogenic in hematopoietic cells. This correlates with the acquisition of cancer hallmarks, such as ‘cytokine-independent survival’, ‘uncontrolled growth’ and ‘genomic instability’. Chromatin dynamics is of pivotal importance for the regulation of transcriptional activity and DNA damage repair. Accordingly, cancer hallmarks are not only effected by oncogenic ‘driver’ alterations at the DNA level, but also at the chromatin level. Sustained DNA binding of constitutively active STAT5 might have distinct effects on chromatin, which might lead to ‘driver’ chromatin alterations and underlie its oncogenicity. The main goal of the present study was to identify ‘driver’ chromatin alterations and other ‘driver’ events during the oncogenesis process induced by constitutively active STAT5. The constitutively active STAT5 mutant STAT5A-1*6 was previously shown to induce oncogenesis in the IL-3-dependent pro-B cell line Ba/F3 by enabling cytokine-independent survival and growth. Specific aims of this study were to characterize the effects of STAT5A-1*6 expression on (i) cell survival and growth, (ii) expression of selected STAT5 target genes and (iii) chromatin rearrangements. To monitor the oncogenesis process, a stable Ba/F3 cell line – inducibly expressing STAT5A-1*6 upon doxycycline administration (Tet-on expression system) – was generated and validated. Short- and long-term STAT5A-1*6 induction experiments were conducted and STAT5A-1*6 protein levels and activation (Western blot), and cell phenotype in terms of survival, growth and genome stability (cell counting, flow cytometric analysis of cellular DNA content/cell cycle states) were analyzed. Expression of STAT5 target genes including Cis, Osm, Spi2.1, c-Myc, Pim-1, Id-1 and TNFRSF13b was investigated in parallel using RT-qPCR. As expected, STAT5A-1*6 expression enabled cytokine-independent survival and growth of Ba/F3 cells. Cell viability and proliferation rates increased gradually during the initial phase of induction. Interestingly, after 4–5 weeks of induction cell survival and growth no longer depended on STAT5A-1*6 expression. In addition, in one out of four experiments, STAT5A-1*6-expressing cells accumulated chromosomal aberrations. The correlation patterns of STAT5A-1*6 and STAT5 target gene expression suggested dose-dependent STAT5A-1*6-mediated transcriptional activation of STAT5 target genes, at least within the first few weeks of induction. Later on however, sustained expression of the STAT5 target genes c-Myc and Pim-1 became independent of STAT5A-1*6. Altogether, these observations suggest the acquisition of the ‘cytokine-independent survival’, ‘uncontrolled growth’ and ‘genomic instability’ cancer hallmarks, possibly due to continually accumulating ‘driver’ alterations upon sustained expression of STAT5A-1*6. Interestingly, using chromatin immunoprecipitation STAT5 DNA binding to Cis, Osm, Spi2.1, Id-1 and TNFRSF13b was correlated with a strong decrease in histone H3 occupancy, likely reflecting a loss in nucleosomes. This histone H3 loss was particularly prominent at the STAT5 binding sites, regardless of (i) their location within the gene locus, of (ii) transcriptional activation and of (iii) cytokine supplementation. In addition, sustained STAT5A-1*6 DNA binding patterns were associated with broadened histone H3 loss in regions distant from STAT5 binding sites. Taken together, these data strongly suggest that DNA binding of STAT5 causes a local nucleosome loss, and possibly a global nucleosome loss along its target genes. Accordingly, I propose a general STAT5-mediated chromatin decondensation mechanism leading to a nucleosome loss around STAT5 binding sites, at a step preceding transcriptional activation. The broadened STAT5A-1*6-associated histone H3 loss patterns also raise the possibility of distinct STAT5A-1*6-mediated ‘driver’ chromatin alterations, which might misregulate chromatin dynamics and, in turn, promote the acquisition of ‘driver’ DNA alterations (i.e. the ‘genomic instability’ cancer hallmark). Together, these DNA and chromatin alteration events might underlie the oncogenicity of constitutively active STAT5. Further characterization of these events might contribute to a better understanding of the mechanism of STAT5-mediated oncogenesis and possibly to the identification of novel molecular targets for the development of drugs against STAT5-associated cancers