---

Medicinal Chemistry

AG Sippl 2022

News

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

More info   

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

New DFG-funded project "Development of fragment-based inhibitors of the bacterial deacetylase LpxC as novel
antibiotics"

The constantly increasing number of multidrug-resistant Gramnegative bacteria poses a pressing threat to human health and welfare. Therefore, novel antibiotics possessing so far unexploited mechanisms of action are urgently required. As lipid A, the hydrophobic membrane anchor of lipopolysaccharides, is essential for growth and viability of Gram-negative bacteria, the inhibition of its biosynthesis represents a promising strategy for the development of antibiotics being selective for Gram-negative germs. The deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis and could be validated as an antibacterial drug target.

In the proposed project Prof. Ralph Holl    (University of Hamburg) and Prof. Wolfgang Sippl (MLU) will develop novel small molecule LpxC inhibitors using innovative fragment-based methods combined with structure-based approaches. NMR-based fragment screening against LpxC will be performed to identify fragments binding to the so far unoccupied UDP-binding pocket of LpxC. Fragment libraries comprising nature-inspired fragments will be screened in the presence of probes being derived from known as well as novel inhibitors.

To generate potent enzyme inhibitors, the identified structures will be merged in the most beneficial way using the knowledge of the structural information derived from experimental NMR data such as Interligand NOEs and molecular docking studies. The envisaged merged compounds will be prepared in a stereocontrolled manner employing state of the art divergent syntheses. Structure-activity relationships will be elaborated for all of the synthesized LpxC inhibitors and rationalized by molecular docking studies. In subsequent optimization steps, the inhibitory activity of the compounds, their antibacterial spectrum against various Gram-negative bacteria as well as their metabolic stability will be improved.

Institute of Pharmacy, May 2020

New textbook "Epigenetic Drug Discovery"

The new book "Epigenetic Drug Discovery" edited by Manfred Jung    and Wolfgang Sippl provides a broad overview of epigenetic approaches in drug research, combining methods and strategies with individual targets. Presented in three parts - Introduction to Epigenetics, General Aspects and Methodologies, and Epigenetic Target Classes - it covers everything any drug researcher would need in order to know about targeting epigenetic mechanisms of disease.

More info here   

Article on novel smHDAC8 inhibitors in ChemMedchem - Front Cover

The Front Cover reflects the structure‐guided optimization of histone deacetylase 8 (smHDAC8) inhibitors active against the human parasiteSchistosoma mansoni. Starting from a cocrystallized fragment‐like hit, structure‐based design and in vitro testing resulted in nanomolar smHDAC8 inhibitors with good selectivity over the predominantly expressed human HDAC1 and 6 isoforms. Four X‐ray structures of smHDAC8 complexes are reported including the one illustrated. The developed inhibitor showed significant, dose‐dependent killing of the schistosome larvae and markedly impaired egg laying of adult worm pairs maintained in culture. More information can be found in the Full Paper by Wolfgang Sippl et al. on page 1517 in Issue 15, 2018 (DOI: 0.1002/cmdc.201800238).   

Histone deacetylases as targets for neuroblastoma therapy

Together with medical chemists from the University of Freiburg as well as the DKFZ research groups "Pediatric Oncology" and "Cancer Drug Development" we developed and tested novel HDAC8 inhibitors for the treatment of neuoblastoma. In additon, ALK kinase was identified as novel target and the combination of HDAC8 inhibitors with the ALK inhibitor crizotinib was found to be significantly more effective than the two inhibitors alone.

see also press release DKFZ Heidelberg   

T. Heimburg et al. (2017) "Structure-Based Design and Biological Characterization of Selective Histone Deacetylase 8 (HDAC8) Inhibitors with Anti-Neuroblastoma Activity". Journal of Medicinal Chemistry, doi:10.1021/acs.jmedchem.7b01447   

J. Shen et al. (2018) “A kinome-wide RNAi screen identifies ALK as a target to sensitize neuroblastoma cells for HDAC8-inhibitor treatment”. Cell Death & Differentiation. doi:10.1038/s41418-018-0080-0   

F. R. Kolbinger et al. (2018). The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines. Arch. Toxicol. 92, 2649-2664, 2018. doi:10.1007/s00204-018-2234-8   

New research project of the research focus program " "Molecular Biosciences as a Motor for a Knowledge-Based Economy"

Development of novel epigenetic inhibitors for the treatment of parasitic diseases

Coworker: Kristin Hausmann

More information can be found here

Article on Protein Degradation Inhibitors - discussed in Science "In the Pipeline"

"In the pipeline" Derek Lowe's commentary on our recently published article "Chemically Induced Degradation of Sirtuin 2 (Sirt2) by a Proteolysis Targeting Chimera (PROTAC) Based on Sirtuin Rearranging Ligands (SirReals)"  in J Med Chem. 2017 Apr 17.   

Prtotein degradation time    . An editorially independent blog from the publishers of Science Translational Medicine.

Bayer PhD Award 2015/16  - Dr. Inna Slynko

Dr. Inna Slynko was awarded with the Bayer PhD price 2015/16. She got the price for her PhD work focusing on “Structural analysis and computer-based design of novel inhibitors for human protein-kinase-C-related kinase PRK1“ . The work was supervised b Prof. Dr. Wolfgang Sippl, Institute of Pharmacy, Halle.

Novel epigenetic inhibitors for the treatment of schistosomiasis

Schistosoma mansoni is a major human pathogen that affects millions of people worldwide and causes  more than 200,000 deaths annually. The fact that there is currently only one drug available for the treatment of schistosomiasis renders urgent the search for new candidates. Lately, drugging the epigenetic machinery of eukaryotic parasites, such as S. mansoni, has become an exciting approach to develop such new antiparasitic drugs.
In previous work, we have shown (Marek et al. PLoS Pathogens 2013) that the histone deacetylase 8 of S. mansoni (smHDAC8) is a promising target for anti-schistosomal drugs. We have identified structural differences in the active site of this lysine deacetylase that can be exploited for species selectivity.

We carried out a structure-based optimization of smHDAC8 inhibitors. Crystallographic analysis provided insights into the inhibition mode of smHDAC8 activity by synthesized 3-amido-benzhydroxamates, where a schistosome-specific clamp plays a pivotal role in the anchoring of the inhibitor molecule in the active site pocket. The newly-designed inhibitors were evaluated in screens for enzyme inhibitory activity against schistosome and human HDACs and in vitro anti-schistosomal activity. The active benzhydroxamates were additionally screened for lethality against the schistosome larval stage (schistosomula) using different assays. Some of the inhibitors showed significant, dose-dependent killing of the schistosome larvae.

In this way, we consider that we have laid the foundations for the development of species selective HDAC8 inhibitors as new antiparasitic drug candidates and chemical probes for dissecting Schistosoma chromatin biology. This may also serve as a model study for other important parasitic pathogens, such as Leishmania or Trypanosoma.

Publication: Heimburg, T. et al. J. Med. Chem. 59(6):2423-35. doi: 10.1021/acs.jmedchem.5b01478   

Histone acetyltransferase inhibitors block neuroblastoma cell growth in vivo

We have previously described novel histone acetyltransferase (HAT) inhibitors that block neuroblastoma cell growth in vitro. Herewe show that two selected pyridoisothiazolone HAT inhibitors, PU139 and PU141, induce cellular histone hypoacetylation andinhibit growth of several neoplastic cell lines originating from different tissues. Broader in vitro selectivity profiling shows thatPU139 blocks the HATs Gcn5, p300/CBP-associated factor (PCAF), CREB (cAMP response element-binding) protein (CBP) and p300,whereas PU141 is selective toward CBP and p300. The pan-inhibitor PU139 triggers caspase-independent cell death in cell culture.Both inhibitors block growth of SK-N-SH neuroblastoma xenografts in mice and the PU139 was shown to synergize withdoxorubicin in vivo. The latter also reduces histone lysine acetylation in vivo at concentrations that block neoplastic xenograftgrowth. This is one of the very few reports on hypoacetylating agents with in vivo anticancer activity.

Full text - Open access:

Oncogenesis (2015) 4, e137; doi:10.1038/oncsis.2014.51   

---

New DFG-funded project "Structure based development and biological characterization of selective inhibitors of histone deacetylases (HDACs) 8 and 10"

The Deutsche Forschungsgemeinschaft (DFG) is funding a new joint project of the Sippl group together with M. Jung (University of Freiburg), I. Oehme and O. Witt (DKFZ Heidelberg) on the optimization of inhibitors of the histone deacetylase (HDAC) subtypes 8 and 10. Histone deacetylases are important modulators of epigenetic gene regulation and the activity of non-histone protein substrates. While for HDACs 1-3 and 6 many potent selective inhibitors have been obtained, for other subtypes much less is known on selective inhibitors and the consequences of their inhibition. In previous work,  HDACs 8 and 10 were identified as promising anticancer targets and respective inhibitors as candidates for further optimization. Crystal structures of HDAC8 and homologues in complex with inhibitors as well as homology models of HDAC10 will be used for structure- and computer-based optimization of the lead structures. Within this project we will further investigate the role of HDAC8 and 10 in the proliferation of cancer cells and optimize available lead structures in a bioguided fashion for potency and selectivity, both in-vitro and in cell culture. Selected improved inhibitors will be subjected to animal studies to clarify the potential of HDAC8 and 10 for future drug development.

---

Meeting Strasbourg

A-ParaDDisE project
Kick-Off meeting, Strasbourg, March 17-18, 2014

The kick-off meeting for the A-ParaDDisE project (EC, FP7-Health) took place in the auditorium of the IGBMC in Illkirch, near Strasbourg on March 17-18. It was hosted by the GIE-CERBM and Dr. Christophe Romier, and the participants were warmly welcomed by the director of the IGBMC (Bertrand Séraphin) and the head of the Structural and Integrative Biology group (Patrick Schultz). Chris Romier gave an introductory presentation of the IGBMC, its history, departments and infrastructures and officially inaugurated the meeting. All participants were unanimous in their praise for the efficient organization of the meeting by the host, which ensured that extremely open and informative discussions could take place. The programme allowed for the presentation of an overview of the project by the coordinator, followed by individual presentations by the project participants and detailed discussions of each Work Package in order to set out strategies for collaborations and material transfers between the teams involved.

---

News archive

Up