Exploring drug interactions at the molecular level

Proteins are an important class of macromolecules: they are essential in a variety of cellular functions, including structural, mechanical, biochemical, and cell signalling processes. To realize these aims, proteins must interact with other biomolecules and small molecule ligands (drugs).

Understanding drug interactions at the molecular level is essential for developing safer and more effective medications.

Combining computational methods, molecular modelling, and experimental data, biotechnologists can explore drug interactions at the molecular level. To achieve this goal, some computational strategies can be followed:

1. Molecular Modelling and Simulation

Molecular modelling involves creating computational models of molecules (such as drugs) and their interactions with target proteins, using supercomputers able to simulate complex molecular systems, including drug-receptor interactions. Techniques like molecular dynamics (MD) simulations allow scientists to explore the dynamic behaviour of molecules over time; for example, researchers can visualize how drugs bind to specific sites on proteins, affecting their function.

2. Drug-Receptor Interactions

A drug’s ability to affect a receptor depends on its “affinity” (the likelihood of occupying a receptor) and “intrinsic efficacy” (how much it activates receptors). These properties are determined by the drug’s chemical structure. Understanding how ligands interact with specific receptors helps predict their effects and design more effective drugs.

3. Molecular Docking

With docking studies, researchers can predict how small molecules (like drugs) or bio-macromolecules interact with target proteins. Various “scoring criteria” are useful for helping predict stable interactions and for designing optimal compounds for the receptor. In this way, it is possible to calculate the orientation and the conformation of docked compounds in the active pockets.

4. Pharmacophore Modelling

This approach represents essential features (steric and electronic) required for optimal interactions with a biological target. By studying drug structure-activity relationships, researchers identify 3D pharmacophore models. These models aid in rational drug design by ensuring effective interactions with specific receptors.

From an experimental point of view, diverse biochemical and biophysical techniques can be used for studying protein-ligand in solution.

In Table 1, you can find a detailed list of the most common and useful methods.

Briefly, X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy provide atomic-resolution or near-atomic-resolution structures of the unbound proteins and the protein-ligand complexes, which can be used to study the changes in structure and/or dynamics between the free and bound forms as well as relevant binding events.

Other experimental approaches that have been applied to study the protein dynamics involved in binding include single-molecule fluorescence spectroscopy and time-resolved hydrogen-deuterium exchange mass spectrometry. Others play an important role in the characterization of protein-ligand binding affinity, such as calorimetry, surface plasmon resonance, and fluorescence.

Table 1. List of the most common and useful methods

A case study: PD and small molecules

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, which affects over 6 million individuals worldwide. PD is characterized by the accumulation of aberrant deposits known as Lewy bodies, which are composed of the aggregated form of the intrinsically disordered protein α-synuclein (Figure 1). Aggregates of α-synuclein, including misfolded oligomers and highly ordered amyloid fibrils, can induce neurotoxicity. However, the precise mechanism is not clarified yet.

Despite significant advances in understanding the molecular basis of PD, there are currently no treatments that can prevent, cure, or significantly delay its progression. Researchers have explored various strategies, including gene editing and cellular therapy, to reduce the toxic α-synuclein aggregates associated with neurodegeneration. However, these approaches can be expensive and challenging to deliver directly into the nervous system.

Figure 1. Aggregates of alpha-synuclein

The intrinsic property of α-synuclein of being unfolded and to appear as an ensemble of energetically similar conformations complicates the rational design of targeted drugs. In fact, it has not been clarified which conformer present in solution is the one responsible for the disease and potentially more toxic (Figure 2).

The incomplete knowledge of these structures makes the development of structure-based drug discovery approaches challenging, as this includes the identification of inhibitors able to prevent α-synuclein aggregation.

Figure 2

Small molecules in PD research

Small molecules and natural products that reduce α-synuclein aggregation levels have become highly sought after. Several drug discovery efforts have analysed large libraries of synthetic compounds to find potential treatments for PD. Some candidates have even progressed to clinical trials. Additionally, the identification of new druggable targets has led to the discovery of small molecules that demonstrate efficacy in pre-clinical studies.

Here are some notable approaches related to small molecules in PD research:

• Stabilizing small molecule blockers: researchers have identified small molecules that target α-synuclein oligomers, able to prevent the misfolding and aggregation of toxic oligomers. Examples are polyphenolic scaffolds, such as natural catecholamines (i.e., dopamine, L-DOPA, epinephrine, and norepinephrine) that can interfere with the aggregation process of α-synuclein. Particularly, dopamine-oxidized derivates redirect the aggregation of α-synuclein to form off-pathway oligomeric structures.
• Repositioning the already approved molecules: Fasudil (Rho-kinase inhibitor and vasodilator), MTC (methylthioninium chloride) and LMTM (leuco-methylthioninium bis-hydromethanesulfonate) have become an attractive strategy for developing novel molecules targeting α-synuclein aggregation. Squalamine and trodusquemine (peptides with antimicrobial activity) have been repositioned for PD due to their potential to prevent α-synuclein lipid-mediated aggregation.
• Rational computational design: new peptidomimetics demonstrate significantly increased blood-brain barrier permeability and have completed clinical Phase I trials, showing promising results in reducing symptoms and improving neuroprotective effects in animal models of PD. High-throughput screening (HTS) identified different compounds that demonstrated effectiveness in animal models of PD.
• Structure-based drug discovery: knowing the three-dimensional structure of the target protein, researchers designed new molecules that specifically bind to and disrupt α-synuclein function. The CryoEM resolution revolution and methodological advances in solid-state NMR offered an unprecedented opportunity to apply this classical approach in the field of amyloids. In fact, recent studies explored atomistic α-synuclein fibril targeted approaches, developing more potent and selective molecules that dock at specific fibril cavities.