Covalent Chemical Cochaperones of the p300/CBP GACKIX Domain

The GACKIX activator binding domain has been a compelling target for small‐molecule probe discovery because of the central role of activator–GACKIX complexes in diseases ranging from leukemia to memory disorders. Additionally, GACKIX is an ideal model to dissect the context‐dependent function of activator–coactivator complexes. However, the dynamic and transient protein–protein interactions (PPIs) formed by GACKIX are difficult targets for small molecules. An additional complication is that activator‐binding motifs, such as GACKIX, are found in multiple coactivators, making specificity difficult to attain. In this study, we demonstrate that the strategy of tethering can be used to rapidly discover highly specific covalent modulators of the dynamic PPIs between activators and coactivators. These serve as both ortho‐ and allosteric modulators, enabling the tunable assembly or disassembly of the activator–coactivator complexes formed between the KIX domain and its cognate activator binding partners MLL and CREB. The molecules maintain their function and selectivity, even in human cell lysates and in bacterial cells, and thus, will ultimately be highly useful probes for cellular studies.     

Small‐Molecule Proteomimetic Inhibitors of the HIF‐1α–p300 Protein–Protein Interaction

The therapeutically relevant hypoxia inducible factor HIF‐1α–p300 protein–protein interaction can be orthosterically inhibited with α‐helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C‐terminal helix of the HIF‐1α C‐TAD (C‐terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side‐chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF‐1α–p300 PPI inhibitors and the first examples of small‐molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF‐1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42‐residue HIF‐1α C‐TAD from which it is derived.     

Substrate Binding Drives Active‐Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot‐Spot Labeling and NMR Spectroscopy Experiments

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between “open”, “semiclosed”, and “closed” conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at “conformational hot spots” of the enzymes. Therefore, we utilized ¹⁵N side‐chain labeling of Trp residues and ¹³C‐methyl labeling of Met residues to study substrate‐induced conformational transitions of GTB. Chemical‐shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical‐shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200–300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB.     

Tampering with Cell Division by Using Small‐Molecule Inhibitors of CDK–CKS Protein Interactions

Cyclin‐dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small‐molecule inhibitors of CDK–CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S‐phase cell populations, and cause a decrease in CDK2, cyclin A, and p27^Kip1 levels. These molecules should help decipher the complex contributions of CDK–CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.     

Kuwanon‐L as a New Allosteric HIV‐1 Integrase Inhibitor: Molecular Modeling and Biological Evaluation

HIV‐1 integrase (IN) active site inhibitors are the latest class of drugs approved for HIV treatment. The selection of IN strand‐transfer drug‐resistant HIV strains in patients supports the development of new agents that are active as allosteric IN inhibitors. Here, a docking‐based virtual screening has been applied to a small library of natural ligands to identify new allosteric IN inhibitors that target the sucrose binding pocket. From theoretical studies, kuwanon‐L emerged as the most promising binder and was thus selected for biological studies. Biochemical studies showed that kuwanon‐L is able to inhibit the HIV‐1 IN catalytic activity in the absence and in the presence of LEDGF/p75 protein, the IN dimerization, and the IN/LEDGF binding. Kuwanon‐L also inhibited HIV‐1 replication in cell cultures. Overall, docking and biochemical results suggest that kuwanon‐L binds to an allosteric binding pocket and can be considered an attractive lead for the development of new allosteric IN antiviral agents.     

Small‐Molecule Inhibitors That Target Protein–Protein Interactions in the RAD51 Family of Recombinases

The development of small molecules that inhibit protein–protein interactions continues to be a challenge in chemical biology and drug discovery. Herein we report the development of indole‐based fragments that bind in a shallow surface pocket of a humanised surrogate of RAD51. RAD51 is an ATP‐dependent recombinase that plays a key role in the repair of double‐strand DNA breaks. It both self‐associates, forming filament structures with DNA, and interacts with the BRCA2 protein through a common “FxxA” tetrapeptide motif. We elaborated previously identified fragment hits that target the FxxA motif site and developed small‐molecule inhibitors that are approximately 500‐fold more potent than the initial fragments. The lead compounds were shown to compete with the BRCA2‐derived Ac‐FHTA‐NH₂ peptide and the self‐association peptide of RAD51, but they had no effect on ATP binding. This study is the first reported elaboration of small‐molecular‐weight fragments against this challenging target.     

Heterotypic Sam–Sam Association between Odin‐Sam1 and Arap3‐Sam: Binding Affinity and Structural Insights

Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTPase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that has high sequence homology with the Sam domain of the EphA2‐receptor (EphA2‐Sam). Both Arap3‐Sam and EphA2‐Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2‐Sam). Recently, we reported a novel interaction between the first Sam domain of Odin (Odin‐Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2‐Sam. In our latest work, we applied NMR spectroscopy, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize the association between Arap3‐Sam and Odin‐Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin‐Sam1 and Arap3‐Sam might bind with a topology that is common to several Sam‐Sam complexes. The revealed structural details form the basis for the design of potential peptide antagonists that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3‐Sam associations.     

Fragmenting the S100B–p53 Interaction: Combined Virtual/Biophysical Screening Approaches to Identify Ligands

S100B contributes to cell proliferation by binding the C terminus of p53 and inhibiting its tumor suppressor function. The use of multiple computational approaches to screen fragment libraries targeting the human S100B–p53 interaction site is reported. This in silico screening led to the identification of 280 novel prospective ligands. NMR spectroscopic experiments revealed specific binding at the p53 interaction site for a set of these compounds and confirmed their potential for further rational optimization. The X‐ray crystal structure determined for one of the binders revealed key intermolecular interactions, thus paving the way for structure‐based ligand optimization.     

Rigidified Clicked Dimeric Ligands for Studying the Dynamics of the PDZ1‐2 Supramodule of PSD‐95

PSD‐95 is a scaffolding protein of the MAGUK protein family, and engages in several vital protein–protein interactions in the brain with its PDZ domains. It has been suggested that PSD‐95 is composed of two supramodules, one of which is the PDZ1‐2 tandem domain. Here we have developed rigidified high‐affinity dimeric ligands that target the PDZ1‐2 supramodule, and established the biophysical parameters of the dynamic PDZ1‐2/ligand interactions. By employing ITC, protein NMR, and stopped‐flow kinetics this study provides a detailed insight into the overall conformational energetics of the interaction between dimeric ligands and tandem PDZ domains. Our findings expand our understanding of the dynamics of PSD‐95 with potential relevance to its biological role in interacting with multivalent receptor complexes and development of novel drugs.     

Design and Generation of Highly Diverse Fluorinated Fragment Libraries and their Efficient Screening with Improved 19F NMR Methodology

Fragment screening performed with ¹⁹F NMR spectroscopy is becoming increasingly popular in drug discovery projects. With this approach, libraries of fluorinated fragments are first screened using the direct‐mode format of the assay. The choice of fluorinated motifs present in the library is fundamental in order to ensure a large coverage of chemical space and local environment of fluorine (LEF). Mono‐ and poly‐fluorinated fragments to be included in the libraries for screening are selected from both in‐house and commercial collections, and those that are ad hoc designed and synthesized. Additional fluorinated motifs to be included in the libraries derive from the fragmentation of compounds in development and launched on the market, and compounds contained in other databases (such as Integrity, PDB and ChEMBL). Complex mixtures of highly diverse fluorine motifs can be rapidly screened and deconvoluted in the same NMR tube with a novel on the fly combined procedure for the identification of the active molecule(s). Issues and problems encountered in the design, generation and screening of diverse fragment libraries of fluorinated compounds with ¹⁹F NMR spectroscopy are analyzed and technical solutions are provided to overcome them. The versatile screening methodology described here can be efficiently applied in laboratories with limited NMR setup and could potentially lead to the increasing role of ¹⁹F NMR in the hit identification and lead optimization phases of drug discovery projects.