Periodic Reporting for period 1 - DUALITY (An Integrated Computational and Experimental Approach to Rapid Synthesis of Highly Selective Dual-Targeted HDAC/CK2, MMP2/CK2 Inhibitors)
Période du rapport: 2018-04-10 au 2020-04-09
Cancer is one of the most important health problems facing our society. It is the second leading cause of death worldwide and was responsible for an estimated 9.6 million deaths in 2018. (https://www.who.int/cancer/PRGlobocanFinal.pdf). Approximately 1 in 6 deaths worldwide are caused by cancer, according to the WHO report (https://gco.iarc.fr/).
Given the complexity of this multifactorial disease, single-target drugs do not achieve satisfactory therapeutic effects. Conversely, drugs directed to more than one target may be clinically advantageous, making multi-target drugs an emerging area of increasing interest to the drug discovery community. This is a working track that we have chosen for this research work.
The targets selected for the discovery of new anticancer drugs are three enzymes involved in the proliferation of tumor processes, namely protein kinase 2 (CK2), Histone deacetylase type1 (HDAC1), and matrix metalloproteinases (MMPs).
Histone deacetylases (HDACs) are a family of epigenetic enzymes that remove acetyl groups from lysine on histones and other proteins. HDAC1 is overexpressed in different human cancers and is regarded as a promising drug target for cancer therapy.
Matrix metalloproteinases (MMPs) are zinc-dependent peptidases involved in the remodeling of the extracellular matrix (ECM). Mammalian MMPs are classified into six subfamilies, one of them (gelatinases) constituted by MMP2 and MMP9. MMP2 has been associated with several pathologies and especially with cancer (https://www.doi.org/10.1039/c3ob41046c). Similarly, Matrix metalloproteinase-13 (MMP-13) slightly promotes tumor invasion and angiogenesis, although it has received the most attention because it is a central node in the cartilage degradation network
The main objective of the project was the development of new multi-target single inhibitors, which might enhance drug efficacy and overcome the current pharmacokinetic limitations.
Previous studies in the host group to obtain MMP2/CK2 dual inhibitors were unsuccessful. However, this study leads to the discovery of a series of highly potent MMP-13 inhibitors. These compounds have utility not only in cancer but mainly in osteoarthritis, which is the single most common cause of disability in older adults in Europe. During the secondment, the researcher studied the drug/ligand interactions of these new interesting inhibitors, using several physicochemical techniques, such as Nuclear Magnetic Resonance (NMR) spectroscopy techniques (Saturation transfer difference (STD and waterLOGSY), Isothermal Titration Calorimetry (ITC), and Surface Plasmon Resonance (SPR) techniques.
A second objective in the project was to conjugate our inhibitors with another moiety capable of binding tumor over-expressed receptors improving their selectivity. After a bibliographic search, this objective was derived from the design of Proteolysis Targeting Chimeras (PROTACS). In this strategy, a protein-ligand is conjugated to a molecule capable of binding ligase (E3), which catalyzes the transfer of ubiquitin to a lysine residue of the target protein promoting its degradation. This technology provides a new attractive approach to drug design. It presents the advantages of being capable to modulate non-traditional drug targets and combat resistance mechanisms.
Given the complexity of this multifactorial disease, single-target drugs do not achieve satisfactory therapeutic effects. Conversely, drugs directed to more than one target may be clinically advantageous, making multi-target drugs an emerging area of increasing interest to the drug discovery community. This is a working track that we have chosen for this research work.
The targets selected for the discovery of new anticancer drugs are three enzymes involved in the proliferation of tumor processes, namely protein kinase 2 (CK2), Histone deacetylase type1 (HDAC1), and matrix metalloproteinases (MMPs).
Histone deacetylases (HDACs) are a family of epigenetic enzymes that remove acetyl groups from lysine on histones and other proteins. HDAC1 is overexpressed in different human cancers and is regarded as a promising drug target for cancer therapy.
Matrix metalloproteinases (MMPs) are zinc-dependent peptidases involved in the remodeling of the extracellular matrix (ECM). Mammalian MMPs are classified into six subfamilies, one of them (gelatinases) constituted by MMP2 and MMP9. MMP2 has been associated with several pathologies and especially with cancer (https://www.doi.org/10.1039/c3ob41046c). Similarly, Matrix metalloproteinase-13 (MMP-13) slightly promotes tumor invasion and angiogenesis, although it has received the most attention because it is a central node in the cartilage degradation network
The main objective of the project was the development of new multi-target single inhibitors, which might enhance drug efficacy and overcome the current pharmacokinetic limitations.
Previous studies in the host group to obtain MMP2/CK2 dual inhibitors were unsuccessful. However, this study leads to the discovery of a series of highly potent MMP-13 inhibitors. These compounds have utility not only in cancer but mainly in osteoarthritis, which is the single most common cause of disability in older adults in Europe. During the secondment, the researcher studied the drug/ligand interactions of these new interesting inhibitors, using several physicochemical techniques, such as Nuclear Magnetic Resonance (NMR) spectroscopy techniques (Saturation transfer difference (STD and waterLOGSY), Isothermal Titration Calorimetry (ITC), and Surface Plasmon Resonance (SPR) techniques.
A second objective in the project was to conjugate our inhibitors with another moiety capable of binding tumor over-expressed receptors improving their selectivity. After a bibliographic search, this objective was derived from the design of Proteolysis Targeting Chimeras (PROTACS). In this strategy, a protein-ligand is conjugated to a molecule capable of binding ligase (E3), which catalyzes the transfer of ubiquitin to a lysine residue of the target protein promoting its degradation. This technology provides a new attractive approach to drug design. It presents the advantages of being capable to modulate non-traditional drug targets and combat resistance mechanisms.
The first step of the project was the selection of the best molecules to be synthesized using computational techniques. The design was based on connecting one group selected to coordinate the Zinc atom present in the active site of HDAC1 (Zinc Binding Group, ZBG), with a moiety based on CX-4945, a potent and selective inhibitor of CK2. The researcher performed an initial High Throughput Virtual Screening (HTVS) to identify the best hits, followed by more precise Glide Docking studies performed with the top hit compounds in order to refine the previously obtained binding mode and to identify the most plausible ligand–CK2/HDAC1 interactions that could account for the molecular recognition process. Thus, we identified the optimal linker length and hydroxamic ZBG as the best moieties to bind the active site of HDAC1 without hampering the binding to CK2 of which the CX4945 moiety was responsible. Molecular dynamics (MD) simulations of the most promising compounds were carried out to assess the stability of the binding modes, and to detect the main molecular interactions that could account for the affinity. Finally, four compounds which presented the higher predicted affinity for both enzymes were selected for synthesis.
The synthesis resulted somewhat difficult, and it was necessary to explore different reaction pathways. Finally, a multistep convergent synthesis allowed us to obtain the final compounds, with the purity necessary (>95%) to carry out biological studies.
Experiments of inhibition in isolated enzymes showed that the synthesized compounds are highly potent and presented balanced activity in both enzymes, an important feature for dual-targeting agents. The best candidate exhibited micromolar activity in cell-based cytotoxic assays against multiple cancer cell lines.
Exploitation and dissemination:
The preliminary results of our systematic investigations have been published in ACS Medicinal Chemistry Letters (https://dx.doi.org/10.1021/acsmedchemlett.9b00561) and a full paper is being prepared for publication
A review article with the title: Molecular Imaging Probes based on Matrix Metalloproteinase Inhibitors (MMPIs) (https://doi.org/10.3390/molecules24162982) has been published in Molecules, an open-access journal,
Another paper is planned to communicate the results obtained for MMP13 inhibitors.
A patent for the protection of the PROTACs for the degradation of CK2 will be applied when the biological tests will be completed.
All the results have been presented in seven national and international meetings, and in several outreach activities.
The synthesis resulted somewhat difficult, and it was necessary to explore different reaction pathways. Finally, a multistep convergent synthesis allowed us to obtain the final compounds, with the purity necessary (>95%) to carry out biological studies.
Experiments of inhibition in isolated enzymes showed that the synthesized compounds are highly potent and presented balanced activity in both enzymes, an important feature for dual-targeting agents. The best candidate exhibited micromolar activity in cell-based cytotoxic assays against multiple cancer cell lines.
Exploitation and dissemination:
The preliminary results of our systematic investigations have been published in ACS Medicinal Chemistry Letters (https://dx.doi.org/10.1021/acsmedchemlett.9b00561) and a full paper is being prepared for publication
A review article with the title: Molecular Imaging Probes based on Matrix Metalloproteinase Inhibitors (MMPIs) (https://doi.org/10.3390/molecules24162982) has been published in Molecules, an open-access journal,
Another paper is planned to communicate the results obtained for MMP13 inhibitors.
A patent for the protection of the PROTACs for the degradation of CK2 will be applied when the biological tests will be completed.
All the results have been presented in seven national and international meetings, and in several outreach activities.
The burden of cancer disease in Europe has been increasing over time. Every year 3.5 million people in the EU are diagnosed with cancer, and 1.3 million die from it. Cancer caused an estimated cost of € 199 billion for society in Europe in 2018.
The potent dual-targeting inhibitors designed and synthesized in this project will contribute to the development of new therapeutic options to address this problem.
The study of MMP13 inhibitors has shed light on the protein/ligand interactions, which will be very useful for the design of the second generation of inhibitors with enhanced selectivity. Osteoarthritis (OA) is the leading cause of joint pain and disability in middle-aged and elderly patients. The clinical utility of broad-spectrum MMP inhibitors developed for the treatment of OA has been restricted by musculoskeletal side effects in humans. Therefore, selectively inhibiting the activity of MMP-13 is expected to have a high impact on the improvement of OA treatments and, consequently, on the quality of life of many EU citizens.
The development of PROTACs is a promising technology in cancer therapy and disease treatment. Our PROTACs will be a starting point for the design of agents to degrade CK2 protein specifically. This unique mode of action, which leads to depletion of the disease target, provides better selectivity compared to classic inhibitors and more sensitivity to drug-resistant targets.
The potent dual-targeting inhibitors designed and synthesized in this project will contribute to the development of new therapeutic options to address this problem.
The study of MMP13 inhibitors has shed light on the protein/ligand interactions, which will be very useful for the design of the second generation of inhibitors with enhanced selectivity. Osteoarthritis (OA) is the leading cause of joint pain and disability in middle-aged and elderly patients. The clinical utility of broad-spectrum MMP inhibitors developed for the treatment of OA has been restricted by musculoskeletal side effects in humans. Therefore, selectively inhibiting the activity of MMP-13 is expected to have a high impact on the improvement of OA treatments and, consequently, on the quality of life of many EU citizens.
The development of PROTACs is a promising technology in cancer therapy and disease treatment. Our PROTACs will be a starting point for the design of agents to degrade CK2 protein specifically. This unique mode of action, which leads to depletion of the disease target, provides better selectivity compared to classic inhibitors and more sensitivity to drug-resistant targets.