The long-term goal of this research is to optimize combinations of quinoline and artemisinin-based drugs to maximize the inhibition of heme crystal formation in the malaria parasite Plasmodium falciparum, particularly in the context of rising drug resistance.

This study investigates a novel, quinoline-like mechanism involving heme-artemisinin adducts. These adducts appear to inhibit heme crystal growth either reversibly or nearly irreversibly, a property directly linked to parasite killing and overcoming resistance mechanisms. Preliminary findings show that heme-artemisinin adducts strongly inhibit heme crystallization and remain effective even against artemisinin-resistant parasites.

To explore this, the research combines laboratory experiments on both sensitive and resistant strains of the parasite, animal model testing, and atomic force microscopy. These methods aim to uncover how drug synergy works at the molecular level and how it influences crystal growth.

The central hypothesis is that heme-artemisinin adducts block heme crystal formation, thereby restoring sensitivity in malaria parasites regardless of their resistance to artemisinin alone. Certain combinations of quinolines, artemisinins, and these adducts may enhance parasite killing by amplifying this inhibition effect.

The study is structured around three specific aims:

1. Determine the inhibitory concentrations and mechanisms of heme-artemisinin adducts in both sensitive and resistant parasites.
2. Assess the reversibility of heme crystal inhibition both in vitro and in vivo.
3. Evaluate how different drug combinations impact parasite killing and the inhibition of heme crystallization.

Overall, this research seeks to develop heme-artemisinin adducts as a potentially safer and more effective antimalarial drug. By targeting hemozoin crystal growth—a validated antimalarial drug target—it also aims to refine drug combination strategies that improve treatment outcomes.

Using a mouse model, the study will help determine which combinations most effectively disrupt crystal growth and lead to parasite death. Ultimately, the findings could support the development of novel therapies against drug-resistant malaria and contribute to safer, more efficient treatment protocols.

Alzheimer’s patients accumulate fibrils and plaques of the protein fragment amyloid-β (Aβ) outside neurons in the brain. Recent clinical evidence strongly supports the amyloid hypothesis, which states that Aβ aggregates impair neurons and promote cognitive decline.

Our main hypothesis is that Aβ fibrils—even if minimally neurotoxic themselves—play a crucial role in generating soluble Aβ oligomers, larger aggregates, or fibril fragments through fragmentation and templating (also called secondary nucleation). All of these species may be neurotoxic.

The assembly of Aβ peptides into fibrils is exceedingly complex, combining peptide chain association from the solution with their restructuring into a conformation typical of the fibril bulk. How incoming peptides fold and bind remains largely unknown, but this step offers new opportunities for rational design of Alzheimer’s disease treatments.

Our approach aims to inhibit amyloidogenesis by elucidating events at the tips of fibrils of different structures (polymorphs) upon the approach of wild-type and mutant peptides, and in the presence of potential fibrillization inhibitors. This will provide fundamental understanding of fibrillogenesis and suggest strategies to suppress fibril formation.

The novelty of our approach is the synergistic combination of molecular dynamics simulations with direct time-resolved imaging using in situ atomic force microscopy (AFM). This allows examination of Aβ fibril growth by monitoring individual fibril elongation.

Correlations between measured growth rates, peptide concentrations, and effects of foreign molecules will illuminate the mechanism of peptide incorporation and yield kinetic and structural parameters for comparison with simulations. Fibril structures will also be characterized by transmission electron microscopy (TEM) to understand their effect on molecular motions.

This three-pronged strategy focuses on crucial events at the fibril tip, avoiding confounding processes such as primary and secondary nucleation involved in bulk aggregation studies.

The research team, with expertise in biological dynamics modeling, protein aggregation thermodynamics and kinetics, and protein assembly structural characterization, will pursue three specific aims:

We will dissect the conformational motions enabling peptide chain incorporation at fibril tips and determine their growth kinetics. Furthermore, we will elucidate how deleterious and protective Aβ mutations, bulk fibril structure, and binding of foreign molecules modulate tip events and fibril growth rates, providing new molecular targets to control fibrillization.

Protein misfolding and subsequent aggregation are major causes of several neurodegenerative diseases, including Alzheimer’s, Parkinson’s, familial amyloid polyneuropathy (FAP), Huntington’s, and type-II diabetes. A common feature in these diseases is the presence of an altered protein form called a partially unfolded amyloidogenic intermediate, which can assemble into harmful amyloid structures.

Recently, scientists have discovered mesoscopic protein-rich clusters that may act as early precursors in the formation of ordered protein solids, such as crystals, sickle hemoglobin polymers, and amyloid fibrils. These clusters challenge traditional views of protein condensation, as the proteins within them display characteristics of both native and partially misfolded forms. Unique properties of these clusters include a kinetically determined size, a thermodynamically controlled number, and a distinct formation pathway compared to other types of protein aggregation, such as those involving disulfide bond reduction.

In our study, we explored how protein structural flexibility influences the formation of these mesoscopic clusters across several proteins. These include:

• p53, a tumor suppressor known as the “guardian of the genome,” which contains multiple disordered and β-sheet-rich domains,
• Hemoglobin A, the main protein in red blood cells, which has a compact, α-helix-rich structure, and
• Lysozyme, an antimicrobial enzyme often used to model protein aggregation.

We found that lysozyme and hemoglobin A form mesoscopic clusters only at high concentrations. In contrast, p53 forms these clusters even at low concentrations and physiological temperatures. This suggests that limited structural flexibility in a protein contributes to its tendency to form such clusters.

Importantly, we observed that crowded environments—like the interior of a cell—promote clustering in intrinsically disordered proteins (IDPs) such as p53. Since around 50% of human cancers involve mutations in p53, this finding is highly relevant. Mutant p53 can act as an oncogene, interfering with normal p53 and other anticancer mechanisms. The tendency of both wild-type and mutant p53 to aggregate is linked to their disease-causing behavior.

Our data show that in crowded environments, p53 clusters can trap surrounding molecules, which creates steric hindrance and increases the energy barrier needed for aggregation. This suggests that the clusters may act as temporary storage for misfolded proteins, potentially giving the cell’s repair systems enough time to refold or degrade them—thereby preventing toxic amyloid formation.

We also found that the nucleation of p53 fibrils does not follow the traditional pathway of individual protein molecules gradually coming together. Instead, the mesoscopic clusters act as pre-assembled, high-concentration sites that accelerate fibril formation. This precursor-based nucleation represents a new biological pathway and opens the door to innovative strategies for preventing protein aggregation in diseases.

We established the mechanisms of action by which several classes of antimalarial drugs and related compounds block hematin crystallization. Research by my colleagues on hematin crystallization, combined with studies of parasite physiology, suggests that hemozoin crystals form within organic sub-phases. These sub-phases consist of a mixture of several lipids, which may be suspended inside the digestive vacuole or line its walls. Their work demonstrates that hematin crystals form via a classical mechanism. New crystal layers nucleate on top of existing layers and grow by the addition of solute molecules, which causes the layers to spread until they cover the entire crystal face.

They also showed that quinoline-class antimalarial drugs effectively inhibit hematin crystallization by binding to specific sites on the crystal surface. This binding blocks the incorporation of soluble hematin, thereby preventing crystal growth.

Crystals represent the most ordered state of matter. The processes by which atoms, molecules, or particles assemble into translationally symmetric arrays have been studied for centuries. However, classical theory predicts slow crystal nucleation rates that diverge significantly from experimental observations. This gap in knowledge presents challenges for controlling crystallization.

Polymorphism is a fundamental property of crystals, where a single compound can adopt multiple crystal structures. This multiplicity results in materials with distinct properties, such as solubility and catalytic activity, which affect their performance across many applications.

Polymorphism can also lead to costly manufacturing challenges. For example, the first HIV/AIDS medication, ritonavir, experienced a shift from a metastable polymorph to a more stable and less soluble form during production, which reduced the drug’s bioavailability. Metastable forms often have higher solubility and may possess other desirable traits like increased compressibility.

Approximately 75% of compounds listed in the Cambridge Structural Database (CSD) crystallize in more than one structure. Crystal polymorphism reflects a free energy landscape (FEL) that, unlike protein folding landscapes optimized by evolution, contains multiple local minima with similar free energies rather than a single global minimum.

The complexity of crystal structures increases further due to their tendency to incorporate solvent molecules, forming solvates.

Our research combines molecular modeling and analytical theory with thermodynamic measurements—such as enthalpies, entropies, and free energies of formation—and kinetic data, including nucleation rates and properties of crystallization precursors. This integrated approach aims to develop:

Ultimately, we will design and validate molecular template-based tools to guide the nucleation of desired polymorphs or to suppress the formation of unwanted crystal forms.