Public defence Olivier Beyens 29/10/2024 - Computational study of DPP8 and DPP9: fundamental insights and inhibitor design - Department Pharamceutical Sciences

Promotors: Prof. Hans De Winter - Prof. Pieter Van der Veken

Location: Aula O1, building O, Campus Drie Eiken

Abstract:

Dipeptidyl peptidyl peptidases (DPP) 8 and 9 are intracellular enzymes with the ability to cleave off a dipeptidyl post-proline from the N-terminal of a substrate. Additionally, DPP9 forms protein-protein complexes with the inflammasome sensors CARD8 and NLRP1. By inhibition of DPP9 the complexes are disrupted and subsequent activation of the pyroptosis pathways occurs. This mechanism leads to cell death in acute myeloid leukemia and HIV-1 cell lines, increasing the interest in DPP9 as a drug target. Research regarding DPP8 is currently in an earlier stage, with less information about the biological function being available.

In this work we focus on a multifaceted computational study on DPP8 and DPP9. In the first objective of this work, we evaluated the structural differences leading to the remarkable selectivity of the DPP9 inhibitor “cpd42” against DPP8. Using an interdisciplinary approach, we validated that the Y280/F253 difference (DPP8/DPP9) is a factor in DPP8 versus DPP9 selectivity of the compound. This result can aid in the further design of selective DPP8/9 inhibitors.

In the second workstream, Hamiltonian Replica Exchange simulations were used to study the R-helix dynamics in DPP9. This R-helix is an α-helix formed by conformational change upon ligand or substrate binding. The R-helix dynamics were compared between a simulation with and without ligand. Removal of the ligand led to a lower occurrence of the R-helix, but even after ligand removal the structured form remained the most prominent conformation of the segment in the simulations. This work can facilitate follow-up research focusing on simulating ligand binding and unbinding pathways to DPP9.

The third objective of this work focusses on the discovery of novel ligands. The chosen methodology to discover new scaffolds was a cosolvent molecular dynamics (MD) informed pharmacophore model. To aid in the setup of cosolvent MD simulations with hydrophobic probes, we developed a new tool called “PART” (Plumed Automatic Restraining Tool), which we released under an open-source license. The resulting cosolvent MD simulations on the targets led to qualitative fragment affinity maps, which were also made freely available. We then used these qualitative fragment affinity maps to perform a pharmacophore screening. Resulting compounds were tested in vitro, but the best compounds were only weak binders.

The final aim focusses on improving the potency of the known DPP8/9 inhibitor (1G244) using relative binding free energy calculations. The optimization of the 1G244 inhibitor led to significant potency improvement, which was validated in vitro.

Public defense Yunita Eka Puspitasari 11/09/2024 - Phytochemical and Antidiabetic Investigations on Marine Natural Products from Indonesia - Department Pharmaceutical Sciences

Promotors:

Prof. dr. N. Hermans (promotor, UAntwerp)

Dr. T. De Bruyne (promotor, UAntwerp)

Prof. dr. A. Aulanni'am (promotor, Universitas Brawijaya)

Prof. dr. ir. A. M. Hariati (promotor, Universitas Brawijaya)

LOCATION: Graduate School Universitas Brawijaya (Indonesia)

Abstract:

Diabetes mellitus is a metabolic disorder characterized by elevated blood glucose levels. Controlling the fluctuation of blood glucose levels by inhibiting α-glucosidase activity is one possible strategy of diabetes management. α-Glucosidase is involved in carbohydrate metabolism by breaking down carbohydrates into their composing monosaccharides such as glucose. Inhibition of α-glucosidase activity slows down the carbohydrate digestion and the subsequent glucose absorption in the intestine.  Marine organisms, such as sea cucumbers and mangrove plants, are important resources of natural products. However, studies on sea cucumbers as a potential source for antidiabetic compounds, are limited. Studies reported that triterpene glycosides have been isolated from the Cuvierian tubules of sea cucumbers. Black sea cucumber (Holothuria atra) however is an exception compared to common sea cucumbers, since it lacks Cuvierian tubules.

In South-East Asia, mangrove (Rhizophora mucronata) bark has been used to treat diabetes, and the most prominent phytochemical compounds thereof, condensed tannins, have been identified. As a result of continuous exploitation of the bark, deforestation of mangrove areas occurs. Bearing this in mind, the leaves, with abundant and faster production, were tested as an alternative diabetes treatment, thus preventing degradation of the marine ecosystem.

This current project was therefore intended to investigate the phytochemical compounds present in the body wall of black sea cucumber (H. atra) and mangrove (R. mucronata) leaves, respectively; and to determine the α-glucosidase inhibitory activity from the body wall of black sea cucumber (H. atra) and mangrove (R. mucronata) leaves by in vitro assays.

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Public defence Natalia Smiejkowska 30/08/2024 - Supporting TB drug discovery through a target-based approach focusing on Mycobacterium tuberculosis mycothione reductase - Department Pharmaceutical Sciences

Promotors: Prof. Yann Sterckx - Prof. Paul Cos

Location:

Aula Q002, gebouw Q

Campus Drie Eiken

Abstract:

Tuberculosis (TB) is an airborne communicable disease that has long been declared a global public health threat. The World Health Organization estimates that a quarter of the world’s population has been infected with Mycobacterium tuberculosis, the etiological agent of TB. Although only 5-10% of infected individuals develop active TB, in 2022, the disease was the second leading cause of death from an infectious agent after COVID-19, with 1.3 million deaths worldwide. Besides its mortality, TB is characterized by a high morbidity, affecting the quality of life and socio-economic development in afflicted regions. A long-term, multidrug treatment and the frequent occurrence of adverse effects complicate patient compliance, leading to an alarming increase in drug resistance, which has become an emerging problem in TB treatment and disease control. There is thus a dire need for the discovery of drugs with novel modes of action and exploration of new drug targets.

M. tuberculosis is an intracellular pathogen that mainly resides in host macrophages. Here, the bacterium encounters dynamic host responses, including the release of reactive oxygen and nitrogen species (ROS and RNS, respectively). Mycothiol (MSH), a low molecular weight thiol, plays a role in maintaining a reducing environment as a key antioxidant. During oxidative stress, MSH acts as a ROS scavenger through its oxidation to mycothiol disulfide (MSSM). The enzyme mycothione reductase (Mtr) recycles MSSM back to MSH, thereby contributing to the maintenance of redox homeostasis.

This thesis explores the early stages of target-based drug discovery, starting from recombinant protein production and purification, through assay development, with the aim to identify and assess the M. tuberculosis Mtr (MtrMtb) inhibitors. The optimisation of MtrMtb production and purification increased protein yield, solubility and stability, thereby ensuring sufficient high-quality material for subsequent stages of the project.

A high-throughput screening (HTS) assay was developed using recombinant MtrMtb, by coupling enzyme activity to a bioluminescent read-out. This assay was applied in a semi-automated setting to screen 137,000 compounds, identifying 19 hits based on their potency, selectivity, and specificity. These hits included two clusters and four fragment molecules, which were further evaluated in whole-cell and intracellular infection assays. Additionally, the role of Mtr in mycobacterial growth, virulence, and stress tolerance was assessed.

The established HTS discovery pipeline offers an opportunity to deliver novel MtrMtb inhibitors and lays the foundation for future efforts in developing robust biochemical assays for the identification and triaging of inhibitors from high-throughput library screens.

Public defence Laurence Van Moll 03/06/2024 - The black soldier fly as a source of new antimicrobials - Department Pharmaceutical Sciences

Promotors:

Prof. dr. Paul Cos (promotor UA)

Prof. dr. Peter Delputte (promotor UA)

Prof. dr. Mik Van Der Borght (promotor KUL)

Prof. dr. Jeroen De Smet (co-promotor KUL)

Location defence:

Aula Q002, gebouw Q

Campus Drie Eiken

Abstract:

With the surge of antimicrobial resistance (AMR), many drugs routinely used in clinical practice are failing to treat critical infectious diseases. Tackling this AMR emergency requires a multifaceted approach, including the discovery and development of new antimicrobial agents that are active against drug-resistant pathogens . Recently, there has been increased interest in natural niches such as insects. Insects are remarkably resilient to microbial infections and possess a wide range of antimicrobial defense tactics. Here, the black soldier fly (BSF, Hermetia Illucens) has been explored as a source of new antimicrobials. A large collection of BSF antimicrobial peptides (AMPs) has been evaluated for its in vitro biological activity using a high-throughput screening platform. Two cecropins (HC1 and HC10) were selected due to their favorable activity profile, being potent action against Gram-negative pathogens and absence of early cytotoxicity indicators. Detailed characterization further elucidated LPS neutralization, membrane permeabilization, fast bactericidal activity, and divalent salt sensitivity. Other characteristics, such as biofilm eradication and synergistic action, were not noted. As these peptides show promising activity against Pseudomonas aeruginosa (with HC10 having confirmed in vivo activity), they can serve as starting points for future antipseudomonal therapeutics. However, some key points of the cecropins, such as their enzymatic stability and biological residency time, remain unaddressed to date. Lastly, this thesis also evaluated the antimicrobial activity of a collection of BSF symbiont strains. The abundant gut isolate Trichosporon asahii showed potent activity against Staphylococcus aureus, but the responsible compounds remain to be investigated.