Peptide-Based Inhibitors of ADAM and ADAMTS Metalloproteinases

ADAM and ADAMTS are two large metalloproteinase families involved in numerous physiological processes, such as shedding of cell-surface protein ectodomains and extra-cellular matrix remodelling. Aberrant expression or dysregulation of ADAMs and ADAMTSs activity has been linked to several pathologies including cancer, inflammatory, neurodegenerative and cardiovascular diseases. Inhibition of ADAM and ADAMTS metalloproteinases have been attempted using various small molecules and protein-based therapeutics, each with their advantages and disadvantages. While most of these molecular formats have already been described in detail elsewhere, this mini review focuses solely on peptide-based inhibitors, an emerging class of therapeutic molecules recently applied against some ADAM and ADAMTS members. We describe both linear and cyclic peptide-based inhibitors which have been developed using different approaches ranging from traditional medicinal chemistry and rational design strategies to novel combinatorial peptide-display technologies

Bacterial Zinc Metalloenzyme Inhibitors: Recent Advances and Future Perspectives

Human deaths caused by Gram-negative bacteria keep rising due to the multidrug resistance (MDR) phenomenon. Therefore, it is a priority to develop novel antibiotics with different mechanisms of action. Several bacterial zinc metalloenzymes are becoming attractive targets since they do not show any similarities with the human endogenous zinc-metalloproteinases. In the last decades, there has been an increasing interest from both industry and academia in developing new inhibitors against those enzymes involved in lipid A biosynthesis, and bacteria nutrition and sporulation, e.g., UDP-[3-O-(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC), thermolysin (TLN), and pseudolysin (PLN). Nevertheless, targeting these bacterial enzymes is harder than expected and the lack of good clinical candidates suggests that more effort is needed. This review gives an overview of bacterial zinc metalloenzyme inhibitors that have been synthesized so far, highlighting the structural features essential for inhibitory activity and the structure-activity relationships. Our discussion may stimulate and help further studies on bacterial zinc metalloenzyme inhibitors as possible novel antibacterial drugs

Hydrolasy závislé na zinku: Studium struktury a funkce glutamátkarboxypeptidasy II a histondeacetylasy 6

Proteiny vázající zinek představují přibližně desetinu proteomu a významnou část z nich tvoří hydrolasy závislé na zinku. Tato disertační práce se zaměřuje na biochemickou a strukturní charakterizaci glutamátkarboxypeptidasy II (GCPII) a histondeacetylasy 6 (HDAC6), které jsou členy rodiny metalohydrolas závislých na zinku, a popisuje interakce s jejich přirozenými substráty a inhibitory. GCPII je homodimerní membránová proteasa, která v centrální a periferní nervové soustavě katalyzuje odštěpení glutamátu z neuropřenašeče N-acetyl-aspartyl glutamátu (NAAG) a v tenkém střevě z folátů přijatých v potravě. Tento enzym je spojován s několika neurologickými poruchami a představuje také ideální cíl pro diagnostiku a léčbu rakoviny prostaty. Inhibitory GCPII obvykle nesou skupinu vázající zinek a dále obsahují funkční skupinu specificky rozpoznávanou v S1' místě enzymu (tvořenou glutamátovým zbytkem nebo jeho isosterem). Tyto sloučeniny jsou přirozeně hydrofilní molekuly, což brání jejich průniku přes hematoencefalickou bariéru a znemožňuje inhibici GCPII v centrální nervové soustavě. V této disertační práci představujeme různé strategie zaměřené na záměnu tradičního P1' glutamátového zbytku nevětvenými neproteinogenními aminokyselinami a bioisostery glutamátu. Dále pak sledujeme vliv zavedení...Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...Katedra biochemieDepartment of BiochemistryFaculty of SciencePřírodovědecká fakult

Mechanism of Calcium-dependent Chloride Channel Activation by the Secreted Regulator CLCA1

The calcium-activated chloride channel regulator (CLCA) proteins are key signaling molecules, which are implicated in various diseases through their tissue-specific expression. Human CLCA1 protein, overexpressed in airway epithelia under pathophysiological conditions, is centrally involved in the manifestation of IL-13-driven mucus cell metaplasia (MCM), a hallmark feature of asthma and chronic obstructive pulmonary disease (COPD), for which there are currently no available therapeutics. Elucidating the poorly understood molecular basis of CLCA1 function is thus required to design specific inhibitors of CLCA1 activity to treat MCM in asthma and COPD. Originally misannotated as ion channels, CLCA proteins are secreted soluble proteins that act as regulators of unknown calcium-activated chloride channels (CaCC) in the airways. Additionally, these proteins undergo proteolytic cleavage, adding yet another level of complexity to the mystery of how a secreted protein can regulate chloride currents. This study focuses on the proteolytic cleavage of CLCA1 and provides mechanistic detail into how secreted CLCA1 protein modulates currents from a CaCC in mammalian cells, which is identified as TMEM16A. For this unique interaction to occur, secreted full-length CLCA1 undergoes a self-cleavage reaction using an N-terminal zincin metalloprotease with a novel fold, which releases the functionally active N-terminal fragment. This cleavage exposes the von Willebrand factor type A (vWA) domain to directly interact with membrane-associated TMEM16A at the C-terminal extracellular loop (loop 9-10). The direct interaction increases TMEM16A surface density by stabilizing the channel on the cell surface and blocking its rapid recycling, thus resulting in increased chloride currents

Proteomic Profiling of Pro and Active Matrix Metalloproteinases using Tandem Mass Spectrometry. Optimization of Affinity Chromatography and nHPLC-MALDI-MS/MS for Proteomic discrimination of Matrix Metalloproteinases in pre-clinical Cancer Model.

Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure.Shaukat Khanam Memorial Cancer Hospital and Research Centre, Pakistan and University of Bradfor

The role of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-­‐15 (ADAMTS-­‐15) in Breast Cancer

Breast cancer is the most common cancer in women and in 2008 accounted for 8% of UK cancer related deaths. A poor prognosis is particularly conferred upon individuals with evidence of metastatic breast cancer. With some studies noting that at least 70% of patients dying with breast cancer have evidence of metastatic disease. In order to develop novel therapeutic strategies a greater understanding of breast cancer tumourigenesis and metastasis is required. Metalloproteinases were implicated as key drivers of metastasis through their ability to degrade the components of the extracellular matrix. This perspective is now superseded with evidence highlighting the involvement of metalloproteinases in an array of biological roles, from maintaining tissue homeostasis to angiogenesis, and importantly these roles can have tumour suppressive effects. Several metalloproteinases from the A Disintegrin and Metalloproteinase with thrombospondin motifs (ADAMTS) family are candidate tumour suppressors, including ADAMTS-15. In the context of breast cancer relatively high levels of ADAMTS-15 expression had previously been associated with increased relapse free survival. However the functional consequences of ADAMTS-15 expression in breast cancer are unknown and are the focus of this thesis. ADAMTS-15 reduced the migration of MDA-MB-231 and MCF-7 cells, in a metalloproteinase-independent manner. This anti-migratory effect likely involves syndecan-4, since modulation of syndecan-4 expression and signalling attenuated this effect. In contrast to its effects on cell migration, only wildtype ADAMTS-15 exhibited an anti-angiogenic effect in in vitro and ex vivo models of angiogenesis. In experimental metastasis assays, both ADAMTS-15 and E362A (metalloproteinase inactive form of ADAMTS-15) reduced metastasis of MDAMB- 231 cells to the liver, though paradoxically, ADAMTS-15 but not E362A enhanced lung colonisation. Taken together these studies demonstrate for the first time that extracellular ADAMTS-15 has multiple tissue context-dependent actions on breast tumour pathophysiology, some of which require its proteolytic activity whereas others do not

System-Level Modeling of Endothelial Permeability Pathway and High-throughput Data Analysis for Disease Biomarker Selection

Ph.DDOCTOR OF PHILOSOPH

Development of Virtual Screening and In Silico Biomarker Identification Model for Pharmaceutical Agents

Ph.DDOCTOR OF PHILOSOPH

DEVELOPMENT OF POLY (2-OXAZOLINE)S-BASED NANOFERROGELS

According to the World Health Organization report, cancer is the second leading cause of death worldwide, and an estimated 10 million deaths were reported due to cancer in 2018. Application of nanotechnology in cancer focuses on development of separate system for diagnosis and treatment of cancer, and it is necessary to shift the paradigm to a combination of diagnosis and treatment on a single platform. Based on the theranostic platform, it may be able to monitor the status of disease in real-time and predict efficacy of treatment. Magnetic nanoparticles have been popular with respect to biomedical applications for diagnosis and cancer treatment. In order to overcome biological challenges of bare magnetic nanoparticles, poly(2-oxazoline)s-based polymeric micelles is designed to load magnetic nanoparticles with anticancer drugs as poly(2-oxazoline)s-based polymers allow facile and broad access to drug delivery. This dissertation research focused on the multiple types of poly (2-oxazoline)-based amphiphilic block copolymers. The effects of various formulation parameters such as (i) polymer and drug structure; (ii) drug and polymer concentration; (iii) environmental factors on the solubilization behavior and physicochemical properties of formulation were explored. A specific poly(2-oxazoline) triblock copolymer was further explored as a vehicle of magnetic nanoparticle-contained theranostic system, NanoFerrogels. The raspberry like-nanoclusters of magnetic nanoparticles were coated by dopamine-decorated polymeric micelles, and paclitaxel (PTX) was encapsulated in the polymeric micelles. Six formulation of PTX loaded-NanoFerrogels were developed depending on (i) molar ratio between magnetic nanoparticles and dopamine-conjugated polymer, (ii) degree of dopamine decoration on polymeric micelles, (iii) PTX loading ratio on polymeric micelles. The release profiles of PTX from NanoFerrogels were dependent on the degree of dopamine conjugation on polymeric micelles. PTX loaded-NanoFerrogels showed similar anticancer activity against several breast cancer cell line compared to free PTX in vitro. PTX loaded-NanoFerrogels significantly triggered release of PTX under alternating current (AC) magnetic field. Another formulational approach of poly(2-oxazoline)-based multifunctional nanocomposite was developed. For co-encapsulating PTX and magnetic nanoparticles, magnetic nanoparticles were coated by oleic acid (MNP-OA). Ferromicelles (PTX-FM) that contained PTX, and MNP-OA by poly(2-oxazoline) polymer can be crosslinked by water soluble linker to form Ferromicelle clusters (PTX-FMC). PTX release from PTX-FMC was triggered when a low frequency AC magnetic field was applied. Cytotoxic effect from PTX-FM, PTX-FMC had similar cytotoxicity compared to PTX encapsulated polymeric micelles against multiple breast cancer cell lines. This dissertation involved the study of remote actuation of NanoFerrogels to cancer cells with low frequency AC magnetic field induced cytoskeletal disruption-based cell death. Furthermore, combination treatment between 18 anticancer drugs and NanoFerrogels under AC magnetic field was screened. Drug selection, dose, and treatment regime were optimized based on the in vitro cytotoxicity studies. In summary, poly(2-oxazoline)-based NanoFerrogels are a promising platform for cancer theranostics.Doctor of Philosoph