Seminar and Practical Course "Drug Target Identification in Tropical Pathogens"

Seminar and Practical Course (SS21), 3 + 3 ECTS

Attendance for the entire module (seminar and practical course) is mandatory.

Seminar in SS 21

The seminar will take place on 10.06., 17.06., 24.06., 1.07., Thursday 17:00-19:00 in online format (via zoom). Zoom link and iCal files will be sent by email to registered participants.

Lecturers for seminar: Michael Boshart, Kristina Malenica, Veronika Ober, Qingping Wu

The assignment of presenters to the dates and papers was done automatically by Excel using a random number generator!

Seminar date	Name of presenter	First name	Primary publication	Lecturer

10. Juni 2021	Schadt	Ella	McNae et al., 2021	Michael Boshart
10. Juni 2021	Qiu	Danni	Wall et al., 2018	Michael Boshart

17. Juni 2021	Linner-Horn	Tamara Petra	Zoltner et al., 2020	Kristina Malenica
17. Juni 2021	Shareef	Riema	Alghamdi et al., 2020	Kristina Malenica

24. Juni 2021	Mahankali	Srujita	Wang et al., 2020	Veronika Ober
24. Juni 2021	NN	NN	Dawidowski et al., 2017	Veronika Ober
24. Juni 2021	Krishna	Krupa	Jain et al., 2017	Veronika Ober

1. Juli 2021	George	Romi Marina	Azad et al. 2021	Qingping Wu
1. Juli 2021	Halder	Hridi	Dolezelová et al. 2018	Qingping Wu

Practical Course in SS 21

online format with focus on computational statistical analysis of small and large datasets and structural analysis of protein-ligand interaction

Topic & aims:

You will get experience on a number of methods including:

in vivo kinase reporter assay for compound screening
in vivo cell viability assay for compound screening
protein-protein interaction analysis using a split-luciferase assay (NanoBiT)
growth curves and protein expression analysis in trypanosomes
computational analysis of protein-ligand interaction using Pymol
quantitative protein-ligand interaction analysis in vitro by Isothermal Titration Calorimetry (ITC)
bioinformatic analysis of kinase-specific proteomics datasets including statistical analysis (Perseus) and database searches (e.g. TriTrypDB, Superfamily, Revigo)

As example we will work on target identification for drug therapy of tropical diseases caused by kinetoplastid parasites (Sleeping sickness, Chagas disease, Leishminiasis). The main research focus of the Boshart lab is protein kinase A (PKA) signaling in Trypanosoma. The parasite PKA qualifies as promising drug target as it is essential for viability and virulence and different from mammalian PKA. In this practical course, we will characterize the ligand specificity of the regulatory T. brucei PKA subunit in vitro and in vivo. Moreover, we will perform in vivo assays for phenotypic characterization.

Trypanosoma brucei is a unicellular, flagellated protozoan parasite belonging to the Kinetoplastida, an evolutionary early branching eukaryotic order. It causes Human African Trypanosomiasis (HAT) and Nagana disease in cattle. The prevalence of these diseases is restricted to sub-Saharan African countries and correlates with the geographical extension of the tsetse fly (Glossina spp.), a blood-sucking insect responsible for transmission of T. brucei. HAT is a severe disease, usually fatal if untreated. The treatment is complicated due to the high costs and the toxicity of some of the available drugs as well as the increasing emergence of drug-resistant trypanosome strains. Hence, there is an urgent need for the identification and validation of new drug targets in these unicellular parasites.

Time schedule of practical work:

The time schedule can be downloaded here: time schedule 2021

Powerpoint presentations:

Introductory Lecture (for download here: https://syncandshare.lrz.de/getlink/fiWf7vQ7qNYwEMLXsViokSuQ/Drug_target_intro_2021.pptx)

Experiments & background week 1

PKA proteome analysis

Deadline for protocol submission is 27.08.21.

Part 1: In vivo studies on T. brucei PKA – cellular and molecular characterization of PKAR mutants

Course script in vivo assays
Files for in vivo analysis

cell count (Excel)

growth curve (Excel)

Western blots PKA expression (zip)

NEB blue prestained protein standard (jpg)

VASP assay Western (zip)

Table 1: Phenotypic in vivo analysis of T. brucei PKAR mutants (Excel)

Alamar Blue analysis (Excel): Toyocamycin, Trypan Blue

Part 2: Assessing T. brucei PKA activity using the NanoBiT assay

This part will be supervised by Qingping Wu, who established this split-luciferase assay in T. brucei during his PhD thesis. Theoretical background as well as a detailed protocol are provided below:

Script and materials of NanoBiT assay (zip)

Part 3: Computational analysis of PKAR-ligand interaction using Pymol and quantitative analysis of PKAR-ligand interaction in vitro by nDSF and ITC

This part will be supervised by Veronica Ober, an expert in structural and computational biology. She will guide you through the Pymol software and help you to recognize critical interactions between protein and ligand.

PyMol script

nDSF&ITC script

Part 4: Statistical analysis and visualization of quantitative proteome data

We will analyze a PKA-dependent quantitative proteome dataset using the software Perseus that was developed by the Max Planck proteomics group. There will be a separate instruction for download from the course webpage how to use the Perseus software. You will get detailed instructions from me on the graphical user interface of this software and the workflow for proteome analysis. The aim is to statistically analyze the comparative quantitative proteome data. The resulting statistically significant protein IDs will be entered into the Trypanosome genome database (TriTrypDB.org), allowing us to identify which molecular pathways may be regulated by PKA.

Skript T brucei proteome analysis - background & protocol

Tutorial: Processing MaxQuant data with Perseus

Proteome data files (zip)

Seminar in SS 20

Topic & aims: The seminar will make you familiar with concepts and methods used in drug development. As an example we focus on drugs for negleted tropical diseases, where academic research plays a particularly important role. The seminar will also provide the background for some of the methods that will be used during the online practical course in SS 20.

Guidelines:

1. Every participant prepares a subject in depth and gives a presentation of 35-40 min + 15-20 min discussion. This presentation reviews the field and provides in depth background on methods relevant for the linked original publication.

2. All participants read the original publications linked to every subject and will be prepared to summarize the scientific questions, approaches, key experiments and conclusions ad hoc in the group discussion of that paper. The person that gives the presentation prepares slides of the figures of the original publication for the group discussion.

3. Literature searches and selection of suitable reviews and materials is up to the presenter. The original publication and some suggestions will be selected by the teaching staff and provided for download in the table below. Participants are encouraged to make an appointment with Dr. Sabine Bachmaier prior to their presentation, where advice on a draft of the presentation and on selected materials can be provided.

Links of interest for preparation:

WHO TDR, DNDI, FAO

Date of Seminar	given by	Time	Subject Title	Suggested Key Paper to be prepared by ALL	Suggested reviews and additional material for preparation of seminar talk	Presentations will be uploaded here after seminar talk (Powerpoint or PDF)
12.05.20	Mahadia Kumkum	13-14:00	Overview on drug discovery for tropical diseases	Field et al., 2017	Rycker et al., 2018	Drug discovery for tropical diseases
12.05.20	Fabian Platten	14-15:00	Phenotypic screening versus target-based drug discovery	Heilker et al., 2019 Aulner et al., 2019	Moffat et al., 2017
15.05.20	Marta Alcala Rey	10-11:00	Protein-protein interactions as drug targets	Scott et al., 2016		Protein-protein interaction as drug targets
15.05.20	Theresa Thanner	11-12:00	Targeting protein-protein interactions in trypanosomes	Dawidowski et al., 2017	Wachsmut et al., 2017 Szöör et al. 2014	Targeting PEX14-PEX5 interaction in T. brucei
19.05.20	Sagarika Ghosh	13-14:00	Phosphodiesterases as drug targets - overview article	Baillie et al., 2019		Phosphodiesterases as drug targets
22.05.20	Iris Lade	10-11:00	Kinases as drug targets	Klaeger et al., 2017		Kinases as drug targets
22.05.20	Sruthi Ramesh	10-11:00	Allostery to disrupt protein kinase interactions	Leroux and Biondi, 2020		Allostery to disrupt protein kinase interactions
30.05.20	Gizem Karabiyik	11-12:00	An unconventional kinase as putative drug target in trypanosomes	Bachmaier et al., 2019		T. brucei protein kinase A as potential drug target
30.05.20	Juliana Giraldo	10-11:00	Chemoinformatics strategies for Leishmaniasis Drug Discovery	Ferreira and Andricopulo, 2018

19.06.20	Dhakad Deepesh	11-12:00	The new drug fexinidazole in a clinical trial	Mesu et al., 2017	Baker and Welburn, 2018 Deeks, 2019
19.06.20	Srishti Ramanathan	12-13:00	Artemisinin - a successful drug against Plasmodium	Zhang et al., 2017	Talman et al., 2019
19.06.20	Ga-Yeon Yoon	13-14:00	Targeting trypanosome PDEs	Blaazer et al., 2018 de Heuvel et al., 2019	https://cordis.europa.eu/project/id/602666/reporting