Andreas C. Joerger

Principles and Applications of Halogen Bonding in Medicinal Chemistry and Chemical Biology

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.

Targeted rescue of a destabilized mutant of p53 by an in silico screened drug

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.

Structural basis for understanding oncogenic p53 mutations and designing rescue drugs

The DNA-binding domain of the tumor suppressor p53 is inactivated by mutation in ≈50% of human cancers. We have solved high-resolution crystal structures of several oncogenic mutants to investigate the structural basis of inactivation and provide information for designing drugs that may rescue inactivated mutants. We found a variety of structural consequences upon mutation: (i) the removal of an essential contact with DNA, (ii) creation of large, water-accessible crevices or hydrophobic internal cavities with no other structural changes but with a large loss of thermodynamic stability, (iii) distortion of the DNA-binding surface, and (iv) alterations to surfaces not directly involved in DNA binding but involved in domain–domain interactions on binding as a tetramer. These findings explain differences in functional properties and associated phenotypes (e.g., temperature sensitivity). Some mutants have the potential of being rescued by a generic stabilizing drug. In addition, a mutation-induced crevice is a potential target site for a mutant-selective stabilizing drug.

The Tumor Suppressor p53: From Structures to Drug Discovery

Even 30 years after its discovery, the tumor suppressor protein p53 is still somewhat of an enigma. p53s intimate and multifaceted role in the cell cycle is mirrored in its equally complex structural biology that is being unraveled only slowly. Here, we discuss key structural aspects of p53 function and its inactivation by oncogenic mutations. Concerted action of folded and intrinsically disordered domains of the highly dynamic p53 protein provides binding promiscuity and specificity, allowing p53 to process a myriad of cellular signals to maintain the integrity of the human genome. Importantly, progress in elucidating the structural biology of p53 and its partner proteins has opened various avenues for structure-guided rescue of p53 function in tumors. These emerging anticancer strategies include targeting mutant-specific lesions on the surface of destabilized cancer mutants with small molecules and selective inhibition of p53s degradative pathways.

Structures of P53 Cancer Mutants and Mechanism of Rescue by Second-Site Suppressor Mutations

We have solved the crystal structures of three oncogenic mutants of the core domain of the human tumor suppressor p53. The mutations were introduced into a stabilized variant. The cancer hot spot mutation R273H simply removes an arginine involved in DNA binding without causing structural distortions in neighboring residues. In contrast, the “structural” oncogenic mutations H168R and R249S induce substantial structural perturbation around the mutation site in the L2 and L3 loops, respectively. H168R is a specific intragenic suppressor mutation for R249S. When both cancer mutations are combined in the same molecule, Arg168 mimics the role of Arg249 in wild type, and the wild type conformation is largely restored in both loops. Our structural and biophysical data provide compelling evidence for the mechanism of rescue of mutant p53 by intragenic suppressor mutations and reveal features by which proteins can adapt to deleterious mutations.

Halogen-Enriched Fragment Libraries as Leads for Drug Rescue of Mutant P53.

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.

Structural evolution of p53, p63, and p73: Implication for heterotetramer formation

Oligomerization of members of the p53 family of transcription factors (p53, p63, and p73) is essential for their distinct functions in cell-cycle control and development. To elucidate the molecular basis for tetramer formation of the various family members, we solved the crystal structure of the human p73 tetramerization domain (residues 351–399). Similarly to the canonical p53 tetramer, p73 forms a tetramer with D2 symmetry that can be described as a dimer of dimers. The most striking difference between the p53 and p73 tetramerization domain is the presence of an additional C-terminal helix in p73. This helix, which is conserved in p63, is essential for stabilizing the overall architecture of the tetramer, as evidenced by the different oligomeric structures observed for a shortened variant lacking this helix. The helices act as clamps, wrapping around the neighboring dimer and holding it in place. In addition, we show by mass spectrometry that the tetramerization domains of p63 and p73, but not p53, fully exchange, with different mixed tetramers present at equilibrium, albeit at a relatively slow rate. Taken together, these data provide intriguing insights into the divergent evolution of the oligomerization domain within the p53 family, from the ancestral p63/p73-like protein toward smaller, less promiscuous monomeric building blocks in human p53, allowing functional separation of the p53 pathway from that of its family members.

Effects of Common Cancer Mutations on Stability and DNA Binding of Full-length p53 Compared with Isolated Core Domains *

Common cancer mutations of p53 tend either to lower the stability or distort the core domain of the protein or weaken its DNA binding affinity. We have previously analyzed in vitro the effects of mutations on the core domain of p53. Here, we extend those measurements to full-length p53, using either the wild-type protein or a biologically active superstable construct that is more amenable to accurate biophysical measurements to assess the possibilities of rescuing different types of mutations by anticancer drugs. The tetrameric full-length proteins had similar apparent melting temperatures to those of the individual domains, and the structural mutations lowered the melting temperature by similar amounts. The thermodynamic stability of tetrameric p53 is thus dictated by its core domain. We determined that the common contact mutation R273H weakened binding to the gadd45 recognition sequence by ∼700-1000 times. Many mutants that have lowered melting temperatures should be good drug targets, although the common R273H mutant binds response elements too weakly for simple rescue.

Small molecule induced reactivation of mutant p53 in cancer cells

The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface crevice that can be targeted by small-molecule stabilizers. Here, we have identified a compound, PK7088, which is active in vitro: PK7088 bound to the mutant with a dissociation constant of 140 μM and raised its melting temperature, and we have determined the binding mode of a close structural analogue by X-ray crystallography. We showed that PK7088 is biologically active in cancer cells carrying the Y220C mutant by a battery of tests. PK7088 increased the amount of folded mutant protein with wild-type conformation, as monitored by immunofluorescence, and restored its transcriptional functions. It induced p53-Y220C-dependent growth inhibition, cell-cycle arrest and apoptosis. Most notably, PK7088 increased the expression levels of p21 and the proapoptotic NOXA protein. PK7088 worked synergistically with Nutlin-3 on up-regulating p21 expression, whereas Nutlin-3 on its own had no effect, consistent with its mechanism of action. PK7088 also restored non-transcriptional apoptotic functions of p53 by triggering nuclear export of BAX to the mitochondria. We suggest a set of criteria for assigning activation of p53.

Toward the Rational Design of P53-Stabilizing Drugs: Probing the Surface of the Oncogenic Y220C Mutant.

The p53 cancer mutation Y220C induces formation of a cavity on the proteins surface that can accommodate stabilizing small molecules. We combined fragment screening and molecular dynamics to assess the druggability of p53-Y220C and map ligand interaction sites within the mutational cavity. Elucidation of the binding mode of fragment hits by crystallography yielded a clear picture of how a drug might dock in the cavity. Simulations that solvate the protein with isopropanol found additional sites that extend the druggable surface. Moreover, structural observations and simulation revealed the dynamic landscape of the cavity, which improves our understanding of the impact of the mutation on p53 stability. This underpins the importance of considering flexibility of the cavity in screening for optimized ligands. Our findings provide a blueprint for the design of effective drugs that rescue p53-Y220C.