Medicinal Chemistry
AG Sippl 2022
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Institut für Pharmazie
Institutsbereich Pharmazeutische Chemie und Klinische Pharmazie
Abteilung Medizinische Chemie
Leiter: Prof. Dr. Wolfgang Sippl
Kurt-Mothes-Straße 3
06120 Halle (Saale)
phone: +49-345-55-25040
fax: +49-345-55-27355
wolfgang.sippl@pharmazie.uni-halle.de
postal address:
Institut für Pharmazie
Institutsbereich Pharmazeutische Chemie und Klinische Pharmazie
Abteilung Medizinische Chemie
06120 Halle (Saale)
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New publication on FLT3-ITD PROTACs in Leukemia
Internal tandem duplications in FMS-like tyrosine kinase-3 (FLT3-ITD) are common mutations in acute myeloid leukemia (AML). In a DFG-funded project, we have now developed two different types of degraders for FLT-ITD and characterized them biologically in leukemia cells. Nanomolar doses of the degraders, a VHL-based PROTAC (MA49) and a degrader with a hydrophobic tag (MA50) induce apoptosis of human leukemic cell lines and primary AML blasts with FLT3-ITD, but not of primary hematopoietic stem cells and differentiated immune cells, FLT3 wild-type cells, retinal cells and c-KIT-dependent cells. The in vivo activity of MA49 against FLT3-ITD-positive leukemia cells was demonstrated in a zebrafish model.
In mechanistic studies, we investigated the effects of the two degraders on different downstram signals. The degrader-induced loss of FLT3-ITD involves the pro-apoptotic BH3-only protein BIM and a previously unidentified degrader-induced depletion of protein folding chaperones. The expression levels of HSP90 and HSP110 correlate with reduced survival of AML patients, and HSP90, HSP110 and BIM are associated with the expression of FLT3 in primary AML cells. HSP90 suppresses degrader-induced FLT3-ITD elimination, establishing a mechanistically defined feedback loop. The two compounds represent novel, potent tools to study the chemical knockout of FLT3-ITD.
New publication in J Med Chem
The NAD+-dependent lysine deacylase sirtuin 2 (Sirt2) is involved in multiple pathological conditions such as cancer. Targeting Sirt2 has thus received an increased interest for therapeutic purposes. In this new publication, we describe the structure-activity studies on 1,2,4-oxadiazole-based analogues, which are potent inhibitors of human Sirt2 deacetylation. As proposed by docking studies, a substrate-competitive and cofactor-noncompetitive binding mode of inhibition could be determined in vitro via binding assays and kinetic analysis and further confirmed by a crystal structure of an oxadiazole inhibitor in complex with hSirt2. Optimized analogues reduced cell viability and inhibited prostate cancer cell migration, in correlation with Sirt2 deacetylase inhibition both in vitro and in cells.
New publication in ACS JCIM
In the current study, cosolvent molecular dynamics simulations were adapted and applied to two Tudor domain-containing proteins, namely Spindlin1 (SPIN1) and survival motor neuron protein (SMN). Tudor domains are characterized by so-called aromatic cages that recognize methylated lysine residues of protein targets. In the study, the conformational transitions from closed to open aromatic cage conformations were investigated by performing MD simulations with cosolvents using six different probe molecules. It is shown that a trajectory clustering approach in combination with volume and atomic distance tracking allows a reasonable discrimination between open and closed aromatic cage conformations and the docking of inhibitors yields very good reproducibility with crystal structures.
First-in-class proteolysis targeting chimera for the Ataxia telangiectasia-and-RAD3-related kinase ATR
About three billion base pairs are replicated within each mammalian cell cycle. Chemotherapeutics kill tumor cells through the induction of DNA replication stress and DNA damage. Exogenous and endogenous DNA stress activate checkpoint kinases, which slow down the cell cycle and initiate DNA repair. The apical checkpoint kinase ataxia telangiectasia-and-RAD3-related (ATR) is activated by stalled DNA replication forks and single strand DNA breaks. Preclinical and clinical studies have demonstrated the efficacy of ATP-competitive ATR inhibitors in combination with chemotherapeutics. Proteolysis-targeting-chimeras (PROTACs) are modern agents that inhibit and eliminate their target proteins by the ubiquitin-proteasome system. We have now developed and characterized the first-in-class PROTAC for ATR in various cell systems. We demonstrate that the cereblon-targeting PROTAC Abd110 decreases ATR dependent on the E3 ubiquitin ligase cereblon and proteasomal activity. Abd110 synergistically induces apoptosis (programmed cell death) of acute myeloid and lymphatic leukemia cells when combined with the clinically used ribonucleotide reductase inhibitor hydroxyurea.
A. M. Alfayomy, R. Ashry, A. Kansy, A.C. Sarnow, F. Erdmann, M. Schmidt, O. H. Krämer, W. Sippl. Design, synthesis, and biological characterization of proteolysis targeting chimera (PROTACs) for the Ataxia telangiectasia and RAD3-related (ATR) kinase. Eur J Med Chem. 267:116167, 2024. doi:10.1016/j.ejmech.2024.116167 .
A. G. Kansy, R. Ashry, A. M. Mustafa, A. Alfayomy, M.P. Radsak, Y. Zeyn, M. Bros, W. Sippl and O. H. Krämer. Pharmacological degradation of ATR induces antiproliferative DNA replication stress in leukemic cells. Mol Oncol. 2024 Mar 22. doi:10.1002/1878-0261.13638.
New DFG Project: "Molecular Design, Synthesis, and Pharmacology of Targeted Protein Degraders for the Checkpoint Kinase ATR"
Kooperation: Prof. Dr. Oliver Krämer, Institut für Toxikologie, Johannes-Gutenbeg Universität Mainz
New publidation: "Novel Nanomolar HDAC Inhibitors and FLT3 inhibition modulate hormesis in leukemic cells with mutant FMS-like tyrosine kinase".
The FMS-like tyrosine kinase-3 (FLT3) is mutated in ~30% of acute myeloid leukemia (AML) patients. Common FLT3 mutations are internal tandem duplications (FLT3-ITD) and point mutations in its c-terminal tyrosine kinase domain (FLT3-TKD). The poor prognosis of patients with FLT3-ITD has spurred an intensive search for FLT3 inhibitors (FLT3i) which showed promising benefits in AML patients with FLT3-ITD.
Epigenetic modifiers of the histone deacetylase (HDAC) family control the development and survival of blood cells. Compared to normal cells, certain leukemic cell types frequently have aberrant expression levels and activities of epigenetic modifiers that belong to the histone deacetylase (HDAC) family. HDACi decrease FLT3-ITD through ubiquitin-dependent proteasomal degradation and apoptosis-associated caspase activation. Combinations of HDACi and FLT3i synergistically kill FLT3-ITD-positive cells through accelerated elimination of FLT3-ITD and DNA replication stress/DNA damage induction. HDAC1, HDAC2, and particularly HDAC3 maintain the stability of FLT3-ITD.
By applying structure based design we were able to develop novel highly potent inhibitors of class I histone deacetylases (HDAC). We reveal that their inhibitory profiles of the lead compounds are superior to those of SAHA and MS-275 in AML cells carrying FLT3-ITD. Low doses of HDACi cause hormesis effects through FLT3-ITD. Specific inhibition of FLT3-ITD with the nanomolar FLT3i marbotinib and quizartinib abrogates undesired hormesis and is synergistically lethal in combination with nanomolar doses of the HDACi KH16. Our finding stresses the need for HDACi that are effective at low nanomolar concentrations. Specific FLT3 kinase inhibitors disable undesired hormesis effects that HDACi cause. This allows such drug combinations to favorably combine against leukemic cells that carry the clinically unfavorable marker FLT3-ITD.
Y. Zeyn, K. Hausmann, M. Beyer, M. Halilovic, H. S. Ibrahim, S. Mahboobi, W. Sippl, O. H. Krämer. Novel Nanomolar HDAC Inhibitors and FLT3 inhibition modulate hormesis in leukemic cells with mutant FMS-like tyrosine kinas. Leukemia, 2023 Sep 21. doi:10.1038/s41375-023-02036-2
Preclinical cancer study: New therapeutic approach for the treatment of neuroblastoma in young children
Neuroblastome are tumors of the nervous system and are the leading cause of cancer-related deaths in young children. A research group at the Universitätsmedizin Halle has now uncovered the processes involved in the development of neuroblastoma for the first time. The protein IGF2BP1 is like a spark that can trigger a whole wildfire of cancer-promoting processes at the cellular level. In preclinical experiments, the researchers used a molecule that could block IGF2BP1 and nip the spark in the bud. During the development of the embryo, the protein IGF2BP1 ensures that cells can grow rapidly. If it occurs later, it is associated with tumors. "In short, IGF2BP1 ensures that another protein is produced more intensively. Both proteins can activate various, previously unexplained processes at the genetic level, which under these flawed circumstances have a strong cancer-promoting effect," explains Sven Hagemann, first author and biochemist at the Institute of Molecular Medicine at the University Medical Center Halle. In cooperation with the Institute of Pharmacy at the Martin-Luther-Universität Halle-Wittenberg , the team has successfully tested a molecule that could block IGF2BP1.
Hagemann S, Misiak D, Bell JL, Fuchs T, Lederer MI, Bley N, Hämmerle M, Ghazy E, Sippl W, Schulte JH, Hüttelmaier S. IGF2BP1 induces neuroblastoma via a druggable feedforward loop with MYCN promoting 17q oncogene expression. Mol Cancer. 2023 May 29;22(1):88. doi:10.1186/s12943-023-01792-0 .
New DFG project in research unit RU5433 RNA in focus
The German Research Foundation is funding a new research group at the Medical Faculty of Martin Luther University Halle-Wittenberg (MLU) with seven million euros. The research focuses on specific RNA molecules and proteins that are probably involved in the formation of tumors. The goal is to understand the underlying mechanisms and thus develop new treatment approaches.
New DFG-funded project "Sirtuin-2 ligands as inhibitors of lysine long-chain deacylation and chemical tools for protein degradation" 2022-2024
Sirtuins are NAD-dependent lysine deacylases that catalyze the cleavage of acetyl but also long-chain acyl groups from lysines in histones and other substrate proteins. This post-translational modification regulates important cellular processes such as metabolism, cell proliferation or migration. The sirtuin isotype Sirtuin2 (Sirt2) has been identified as a target for drug discovery in the areas of oncology, inflammation and neurodegeneration. Dual inhibition of both deacetylation and deacylation has been postulated to be highly effective for potential anticancer drugs. In preliminary work, we have developed new lead structures for potent and selective Sirt2 inhibitors that exhibit this dual inhibition profile and block prostate cancer cell migration better than mere acetylation inhibitors. In addition, we have discovered selective inhibitors of long-chain deacylation that are unique to date. In this project, we aim to optimize the potency and cellular efficacy of dual acetylation/deacylation and selective deacylation inhibitors to further unravel the role of the different hydrolase activities of Sirt2 and exploit their therapeutic potential. In addition to "classical" inhibitors, we further intend to develop novel proteolysis-inducing chimeras (PROTACs) for Sirt2 as an orthogonal approach. These will be hybrid inhibitors that simultaneously target Sirt2 and an appropriate E3 ligase. This will lead to ubiquitinylation of Sirt2 and subsequent proteasomal degradation, which will also lead to a dual activity profile. In addition to validated ligases such as Cereblon and VHL, we will develop new ligands for the ligase Parkin, which we have discovered to be suitable for Sirt2 degradation, thus expanding the available arsenal for Sirt2 PROTACs and PROTACs in general.
Collaboration partner: Prof. Manfred Jung, Albert-Ludwigs-University of Freiburg
New publication in Nature Chem. Biology - Profiling of HDAC Inhibitors by Chemical Proteomics
Mass-spectrometry based proteomics is the big-data science of proteins that allows the monitoring of the abundance of thousands of proteins in a sample at once. Therefore, it is a particularly well-suited readout for discovering which proteins are targeted by any small molecule. An international research team including the MedChem grup at the MLU Halle-Wittenberg has investigated this using chemical proteomics.
Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target . In: Nature Chemical Biology. DOI: 10.1038/s41589-022-01015-5
New DFG-funded project "Synthesis and pharmacology of novel inhibitors of histone deacetylases and of proteolysis targeting chimeras (PROTACs) for mutant FMS-like tyrosine kinase-3" 2022-2024
Acute myeloid leukemias (AML) with mutations in the kinase FMS-like tyrosine kinase-3 (FLT3) are a clinically unsolved problem. The most common mutations of FLT3 associated with AML are in its juxtamembrane domain (FLT3-ITD). Quite a few of the existing FLT3 inhibitors are very potent. However, they are not very effective against mutations in the FLT3 tyrosine kinase domain (FLT3-TKD) that arise during the therapy with such inhibitors, or they are not specific for FLT3. A major goal in the development of new FLT3 inhibitors is to identify a molecule that inhibits mutant FLT3 in the DFG-in and DFG-out conformations in a highly potent manner, while not acting against kinases that are necessary for normal hematopoiesis. Since inhibitors of the histone deacetylase (HDAC) family promote the degradation of mutant FLT3, we synthesized and tested novel inhibitors of this class of compounds. These epigenetic modulators specifically inhibit the tumor-relevant class I HDACs (HDAC1, HDAC2, HDAC3) and are more selective and effective than clinically tested class I HDAC inhibitors against AML cells with FLT3-ITD and FLT3-TKD mutants. Other available, structurally related HDAC inhibitors will be analyzed for their effects against permanent and primary leukemia cells with mutated FLT3. In this context, we aim to molecularly explain our unexpected observation on a dose-dependent switch from a stabilization to a degradation of FLT3. In addition, we have developed and tested protein-degrading inhibitors (so-called PROTACs, which cause proteasomal degradation of their target proteins) for FLT3. We have discovered for the first time a potent mutant-specific PROTAC for FLT3-ITD and FLT3-TKD, which we now aim to optimize with respect to its biological effects on leukemia cells. This will be done using structure-based optimization, innovative PROTAC synthesis concepts, in vitro inhibition/selectivity assays, and cellular characterization. We aim to link the best FLT3-ITD inhibitor scaffolds (scaffolds) with different ubiqutin E3 ligase ligands to create even better PROTACs. As a further goal, we aim to test and molecularly understand the anti-leukemic effects of FLT3 PROTACs alone and in combination with novel HDACi in permanent and primary AML cells. We will employ modern synthetic approaches, global transcriptome, proteome and phospho-proteome analyses, individual and kinome-wide selectivity studies, targeted protein analyses, flow cytometry and genetic knockout strategies. This will advance the preclinical establishment of HDAC inhibitors and FLT3 PROTACs and we can provide evidence for innovative, rationally designed combination therapies.
Collaboration partner: Prof. Oliver Krämer , Institut für Toxikologie und Pharmakologie, Johannes-Gutenbeg Universität Mainz. Prof. Mike Schutkowski, Institut für Biochemie, MLU Halle-Wittenberg
New DFG-funded project "Molecular Design, Synthesis, and Pharmacology of Novel and Selective HDAC10 Inhibitors" 2022-2024
Histone deacetylases (HDACs) are a family of 18 epigenetic modifiers that fall into 4 classes. Histone deacetylase inhibitors (HDACi) are developed to correct dysregulated biological processes due to aberrant HDAC activities. HDAC6 and HDAC10 belong to the class IIb subgroup of the HDAC family. The targets and biological functions of HDAC10 are still ill-defined and no specific HDAC10 inhibitors with biological activity are published. We have synthesized and characterized the first specific inhibitors of HDAC10 and we demonstrate that these induce apoptosis of acute B cell leukemia and lymphoma cells. We aim to rationally improve our lead compounds by a combination of structure-based design, synthesis, in vitro testing as well as cellular characterization including genetic knockout strategies. Promising candidates can be selected based on the in vitro profiling using recombinant HDACs. We will test such inhibitors against a larger panel of leukemic cells regarding apoptosis induction. To comprehensively reveal the protein targets of HDAC10, we will use our new HDAC10 inhibitors in global proteome and transcriptome analyses. This will include a comparative analysis of leukemic cells that respond or resist apoptosis induction upon HDAC10 inhibition and we want to carry out functionally tests for new HDAC10 targets. To discover further innovative inhibitors of HDAC10, we will synthesize and test proteolysis targeting chimeras (PROTACs) that inhibit the catalytic activity of HDAC10 and additionally degrade it. Our new HDAC10 inhibitors are tools to identify the downstream targets and functions of HDAC10. Moreover, such compounds could prospectively be used as new treatment options for leukemia.
Collaboration partner: Prof. Oliver Krämer , Institut für Toxikologie und Pharmakologie, Johannes-Gutenbeg Universität Mainz. Prof. Mike Schutkowski, Institut für Biochemie, MLU Halle-Wittenberg
New DFG/ANR-funded project on the development of novel anti-malaria drug leads (DualTargApi) 2022-2024
Plasmodium falciparum, a protozoan pathogen, is still the greatest threat causing malaria with severe clinical significance and negative socio economic impact. This parasite possesses a substantial repertoire of conserved enzymes including those involved in chromatin remodeling and histone modifications. These enzymes have been described to play vital roles in epigenetic mechanisms for spatio-temporal regulation of gene expression that are crucial for parasite growth and differentiation. For instance, histone deacetylases (HDAC), histone acetyltransferases (HAT) and methyltransferases (HMT) play key roles in cell cycle progression, and particularly in the control of variable surface gene expression involved in immune evasion by the parasite. These enzymes are therefore considered as valid therapeutic targets. We started testing this hypothesis and reported the development of novel HDAC inhibitors against P. falciparum. More recently, preliminary data with dual-targeting compounds that were designed by fusing another inhibitor scaffold with HDAC inhibitors showed potent antiplasmodial activity against resistant P. falciparum strains. The synergy of inhibiting both targets was confirmed in a combination assay where the combination of two individual drugs showed potent inhibition of the parasite growth (Pf 3D7) at low concentration. In the present project, we will exploit the basic principles and major results to target P. falciparum and develop the inhibitors into potent, selective and in vivo active drug candidates. This will lead to the generation of novel hybrid antiparasitic compounds with a high potential to delay or circumvent the development of resistance and the ability to provide additional range of treatments with effective combination options. In parallel, we propose to define the mode of action of the most promising dual-targeting compounds that will give rise to new approaches for examining and manipulating biological processes and will enhance the understanding of how PfHDAC enzymes work. This could be achieved through the chemistry/biology cross fertilization and the generated knowledge will likely continue to improve the quality of treatments against apicomplexans.
Keywords: Plasmodium, histone deacetylase, drug design, dual-targeting inhibitors, neglected tropical diseases.
Collaboration partner: Jamal Khalife (PhD), The Institute for Infection and Immunity, Institut Pasteur Lillle, France