Halogen bonding has been known in material science for decades, but until recently, halogen bonds in protein–ligand interactions were largely the result of serendipitous discovery rather than rational design. In this Perspective, we provide insights into the phenomenon of halogen bonding, with special focus on its role in drug discovery. We summarize the theoretical background defining its strength and directionality, provide a systematic analysis of its occurrence and interaction geometries in protein–ligand complexes, and give recent examples where halogen bonding has been successfully harnessed for lead identification and optimization. In light of these data, we discuss the potential and limitations of exploiting halogen bonds for molecular recognition and rational drug design.

The tumor suppressor p53 is mutationally inactivated in ≈50% of human cancers. Approximately one-third of the mutations lower the melting temperature of the protein, leading to its rapid denaturation. Small molecules that bind to those mutants and stabilize them could be effective anticancer drugs. The mutation Y220C, which occurs in ≈75,000 new cancer cases per annum, creates a surface cavity that destabilizes the protein by 4 kcal/mol, at a site that is not functional. We have designed a series of binding molecules from an in silico analysis of the crystal structure using virtual screening and rational drug design. One of them, a carbazole derivative (PhiKan083), binds to the cavity with a dissociation constant of ≈150 μM. It raises the melting temperature of the mutant and slows down its rate of denaturation. We have solved the crystal structure of the protein–PhiKan083 complex at 1.5-Å resolution. The structure implicates key interactions between the protein and ligand and conformational changes that occur on binding, which will provide a basis for lead optimization. The Y220C mutant is an excellent “druggable” target for developing and testing novel anticancer drugs based on protein stabilization. We point out some general principles in relationships between binding constants, raising of melting temperatures, and increase of protein half-lives by stabilizing ligands.

The destabilizing p53 cancer mutation Y220C creates a druggable surface crevice. We developed a strategy exploiting halogen bonding for lead discovery to stabilize the mutant with small molecules. We designed halogen-enriched fragment libraries (HEFLibs) as starting points to complement classical approaches. From screening of HEFLibs and subsequent structure-guided design, we developed substituted 2-(aminomethyl)-4-ethynyl-6-iodophenols as p53-Y220C stabilizers. Crystal structures of their complexes highlight two key features: (i) a central scaffold with a robust binding mode anchored by halogen bonding of an iodine with a main-chain carbonyl and (ii) an acetylene linker, enabling the targeting of an additional subsite in the crevice. The best binders showed induction of apoptosis in a human cancer cell line with homozygous Y220C mutation. Our structural and biophysical data suggest a more widespread applicability of HEFLibs in drug discovery.

Aggregation of destabilized mutants of the tumor suppressor p53 is a major route for its loss of activity. In order to assay drugs that inhibit aggregation of p53, we established the basic kinetics of aggregation of its core domain, using the mutant Y220C that has a mutation-induced, druggable cavity. Aggregation monitored by light scattering followed lag kinetics. Electron microscopy revealed the formation of small aggregates that subsequently grew to larger amorphous aggregates. The kinetics of aggregation produced surprising results: progress curves followed either by the binding of Thioflavin T or the fluorescence of the protein at 340 nm fitted well to simple two-step sequential first-order lag kinetics with rate constants k ₁ and k ₂ that were independent of protein concentration, and not to classical nucleation-growth. We suggest a mechanism of first-order formation of an aggregation competent state as being rate determining followed by rapid polymerization with the higher order kinetics. By measuring the inhibition kinetics of k ₁ and k ₂ , we resolved that the process with the higher rate constant followed that of the lower. Further, there was only partial inhibition of k ₁ and k ₂ , which showed two parallel pathways of aggregation, one via a state that requires unfolding of the protein and the other of partial unfolding with the ligand still bound. Inhibition kinetics of ligands provides a useful tool for probing an aggregation mechanism.