Affinity, Gel Filtration, Size Exclusion Chromatography: Repurposing Kinase Inhibitors for Malaria

Join us for a fascinating conversation with Dr. Subhoja Chakraborty from the University of Central Florida, whose cutting-edge research targets the global challenge of malaria drug resistance. Dr. Chakraborty shares her journey from structure-based drug design during her PhD to her recent collaborative work repurposing human kinase inhibitors as rapid, selective antimalarials against drug-resistant Plasmodium falciparum strains.

In this episode, you'll learn:

• How structure-guided approaches reveal potential drug targets, including cysteine proteases like falcipain-2, and kinases implicated in malaria's complex life cycle.
• The power of repurposing kinase inhibitors originally developed for cancer and other diseases, focusing on compound 12, which blocks hemozoin formation and protein kinase 6—key steps in parasite survival.
• Lab techniques such as affinity, gel filtration, and size exclusion chromatography for recombinant protein purification—and how troubleshooting is crucial for high-yield, stable complexes.
• Advances in mass spectrometry for characterizing protein interactions, validating drug targets, and mapping molecular pathways pivotal to malaria therapeutics.
• Tips and lessons for new researchers facing the challenges of sample preparation, complex protocols, and working as part of a collaborative team.

Dr. Chakraborty’s insights bridge the gap between basic science and translational drug discovery, offering hope for more effective, targeted malaria treatments in a world where resistance to frontline therapies threatens millions.

Referenced Papers:

• "Plasmodium falciparum protein kinase 6 and hemozoin formation are inhibited by a type II human kinase inhibitor exhibiting antimalarial activity" (Cell Chemical Biology, 2025)
• "New insights of falcipain 2 structure from Plasmodium falciparum 3D7 strain" (BBRC, 2022)
• "Structure-Based Optimization of Protease−Inhibitor Interactions to Enhance Specificity of Human Stefin‑A against Falcipain‑2 from the Plasmodium falciparum 3D7 Strain" (Biochemistry, 2023)

If you’re a student, scientist, or simply passionate about advances in infectious disease research, this episode offers actionable insights, inspiring advice, and a front-row seat to innovation in combating malaria.

Video Transcription

From Structural Biology to Drug Discovery: Chromatography and Mass Spectrometry in Malaria Research

Introduction

In this discussion, Subhoja shares insights into her research journey, focusing on malaria drug discovery and the role of modern analytical techniques in biological and biomedical research. The conversation is aimed primarily at undergraduate students and early-career researchers interested in chemistry, biotechnology, and life sciences.

Research Focus: Malaria Drug Discovery and Kinase Targets

Subhoja is currently involved in malaria drug discovery research at the University of Central Florida, where she has been working since beginning her postdoctoral position two years ago. Her earlier PhD research focused on structure-based drug design in malaria, using a combination of in silico approaches, recombinant protein expression, and X-ray crystallography to determine precise three-dimensional structures of target proteins and their inhibitors.

This structural perspective provided a foundation for her current work, which centers on drug discovery through compound screening and repurposing. A recent publication from her laboratory explores the use of repurposed mammalian CDK-like kinase inhibitors as potential antimalarial agents. While such kinase inhibitors are well known in cancer and cardiovascular research, their application to malaria targets represents a novel and promising approach.

Kinases are attractive drug targets because of their central role in cell signaling and growth pathways. By targeting specific kinases involved in different stages of the malaria parasite life cycle—whether asexual growth, gametocytogenesis, or differentiation—it may be possible to significantly inhibit parasite growth and progression.

Chromatography in Protein Expression and Purification Workflows

Chromatography plays a critical role throughout Subhoja’s research. Her experience spans from basic chromatography techniques during her early training to advanced protein purification workflows using affinity chromatography, gel filtration (size exclusion chromatography), and ion exchange chromatography.

Recombinant proteins are routinely purified using affinity resins such as nickel or cobalt, followed by further purification to remove contaminants. Fast Protein Liquid Chromatography (FPLC) enables compact, stepwise purification workflows suitable for producing proteins for in vitro assays.

A recurring challenge in this process is the expression of high-molecular-weight proteins, which often accumulate in insoluble fractions or inclusion bodies. Careful selection of expression vectors, host systems, and induction conditions is therefore essential to maximize yield and solubility.

Integrating Size Exclusion Chromatography into the Workflow

Size exclusion chromatography (SEC) is typically used after affinity purification to further improve protein purity. Following elution with imidazole or glutathione, desalting is required to prevent buffer components from interfering with downstream assays. This is often achieved using desalting columns such as G-25 or PD-10.

To remove lower-molecular-weight contaminants, finer SEC media—such as Superdex or Sephacryl columns—are employed. Concentration steps using molecular-weight cut-off filters are then applied to obtain the desired protein concentration for functional studies.

These iterative purification and troubleshooting steps are a routine part of protein biochemistry and reflect the importance of methodical optimization and careful validation.

The Role of Mass Spectrometry in Biomolecular Characterization

Mass spectrometry is a key analytical tool in Subhoja’s research pipeline, particularly for target identification and validation. In both malaria and cancer research, MS is used alongside chromatography, immunoprecipitation, and pull-down assays to study protein interactions and molecular recognition events.

Affinity-based mass spectrometry enables the identification of binding partners and interactors, whether studying proteins, long non-coding RNAs, or protein complexes. Techniques such as native MS, tandem affinity purification, and proximity labeling (e.g., TurboID-based workflows) provide deeper insights into molecular networks and signaling pathways.

These approaches are essential for understanding how candidate drug targets function within complex biological systems.

Sample Preparation Challenges in Biological Research

Sample preparation remains one of the most challenging aspects of biological experimentation. Whole-cell lysates contain proteases, nucleases, and other components that can rapidly degrade target molecules. Preventing degradation requires minimizing preparation time, maintaining low temperatures, and using appropriate inhibitor cocktails.

Reproducibility is also critical: biological experiments require multiple replicates, and variability is often unavoidable. Careful handling, protocol optimization, and continuous troubleshooting are essential to ensure reliable results, whether preparing samples for chromatography, mass spectrometry, sequencing, or immunoprecipitation.

Advances in Chromatography and MS for Malaria Research

Modern advances in chromatography and mass spectrometry significantly enhance malaria research by enabling precise target identification, quantification, and functional characterization. Drug discovery workflows begin with construct design and recombinant expression, followed by purification and interaction studies.

In malaria research, stage-specific parasite biology requires careful extraction and analysis of proteins from different life cycle stages. Tandem affinity MS, immunoprecipitation, and in vivo validation studies help confirm target relevance and therapeutic potential.

Recent work on repurposed kinase inhibitors has shown promising results, including inhibition of hemozoin formation—a key detoxification pathway for the malaria parasite—highlighting the potential of such compounds for further development.

Malaria, Drug Resistance, and Global Impact

Malaria remains a major global health burden, with over 600,000 deaths annually and widespread drug resistance complicating treatment. Artemisinin resistance, particularly associated with mutations in the K13 propeller domain, has driven the search for new targets and therapies.

During her PhD, Subhoja focused on falcipain-2, a cysteine protease involved in hemoglobin degradation within the parasite. This pathway is critical for parasite survival and has emerged as a promising drug target. Her work revealed that falcipain-2 operates within a larger protein complex responsible for hemoglobin processing and amino acid release.

Understanding these molecular mechanisms is essential for developing robust, stage-specific antimalarial strategies.

Advice for Early-Career Researchers

For researchers just starting out, Subhoja emphasizes the importance of hands-on experience. Working directly with cells, lysates, and analytical tools is essential for truly understanding experimental systems. Failure is an inherent part of research, and perseverance is key.

Motivation, teamwork, careful sample handling, and thorough preparation all contribute to long-term success. Reading extensively, critically evaluating one’s own work, and appreciating incremental progress are vital aspects of a sustainable research career.

Every successful experiment—no matter how small—represents meaningful progress and contributes to the broader goals of science and medicine.

Closing Remarks

The discussion highlights how interdisciplinary approaches combining chromatography, mass spectrometry, structural biology, and biochemistry are driving advances in malaria drug discovery. Through careful experimental design, analytical rigor, and collaborative teamwork, researchers can address complex biological challenges and contribute to global health solutions.

This text has been automatically transcribed from a video presentation using AI technology. It may contain inaccuracies and is not guaranteed to be 100% correct.

Concentrating on Chromatography Podcast

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