Synthetic Inhibitors of Extended Helix–Protein Interactions Based on a Biphenyl 4,4′‐Dicarboxamide Scaffold

Turn Bak : We present rationally designed scaffolds that mimic the spatial projection of the i, i+4, i+7, and i+11 residues of an α‐helix. A library of biphenyl derivatives was shown by competition fluorescence polarization and ITC to mimic Bak and disrupt the Bak/Bcl‐x_L protein–protein interaction. ¹⁵N HSQC experiments confirmed that the surface of Bcl‐x_L normally occupied by Bak was the target area of our new synthetic inhibitors.

     

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.     

Site‐specific Protein Cross‐Linking with Genetically Incorporated 3,4‐Dihydroxy‐L‐Phenylalanine

Come together right now with L ‐DOPA : Chemical cross‐linking is widely used to study protein–protein interactions. However, many cross‐linking agents suffer from low reactivity or selectivity. An efficient and selective reaction of site‐specific protein cross‐linking was achieved using genetically incorporated 3,4‐dihydroxy‐L ‐phenylalanine.

     

Computational Studies Identifying Entry Inhibitor Scaffolds Targeting the Phe 43 Cavity of HIV‐1 gp120

Targeting protein–protein interactions, such as the HIV‐1 gp120—CD4 interface, has become a cutting‐edge approach in the current drug discovery scenario. Many small molecules have been developed so far as inhibitors of the interaction between CD4 and HIV‐1 gp120. However, due to a variety of reasons such as solubility, drug toxicity and drug resistance, these inhibitors have failed to prove clinically useful. As such, the identification of novel compounds that bind to protein–protein interactions is still a research area of considerable interest. Here, a structure‐based virtual screening approach was successfully applied with the aim of identifying novel HIV‐1 entry inhibitors targeting the Phe 43 pocket of HIV‐1 gp120. Several compounds able to inhibit viral replication in cell culture were identified, with the best agent endowed with an EC₅₀ value of 0.9 μM . Inactivity of all the identified hits toward a mutant (Met 475 Ile) strain strongly suggests that they interact in the Phe 43 cavity of gp120, as intended. Remarkably, all of these small molecules have a chemical scaffold unrelated to any known class of entry inhibitors reported thus far. Overall, our strategy led to the identification of four novel chemical scaffolds that inhibit HIV‐1 replication through the destabilization of the HIV‐1 gp120–CD4 interface.     

Tailor‐Made Designer Helical Peptides that Induce Mitochondrion‐Mediated Cell Death without Necrosis

Managing protein–protein interactions is essential for resolving unknown biological events at the molecular level and developing drugs. We have designed and synthesized a side‐chain‐crosslinked helical peptides based on the binding domain of a pro‐apoptotic protein (Bad) that induces programed cell death. The peptide showed high helical content and bound to its target, Bcl‐X_L, more strongly than its non‐crosslinked counterparts. When HeLa cells were incubated with the crosslinked peptide, the peptide entered the cytosol across the plasma membrane. The peptide formed a stable complex with Bcl‐X_L localized at the outer mitochondrial membrane, and this binding event caused the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. This activated the caspase cascade: 70 % of HeLa cells died by the apoptosis pathway (without evidence of necrosis).     

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.     

Foldamers as Anticancer Therapeutics: Targeting Protein–Protein Interactions and the Cell Membrane

Targeting important protein–protein interactions involved in carcinogenesis or targeting the cell membrane of a cancer cell directly are just two of the ways in which foldamers (oligomeric molecules that fold into distinct shapes in solution) hold considerable potential in the treatment of cancer. From mimicking the local topography of the helical compound of interest by using covalently constrained foldamers to mimicking the topography of the natural helix such that the positions of key functional motifs are in an identical spatial orientation to match those presented by the original α‐helix, synthetic foldamers have been used to mimic the natural foldamers that interact with proteins or the cell membrane. These targeted approaches have become established over a timeframe of more than a decade, and they continue to be included in the assortment of cancer targets being studied and the arsenal of chemotherapy compounds in development. These approaches are reviewed herein.     

Rational Design,Binding Studies,and Crystal‐Structure Evaluation of the First Ligand Targeting the Dimerization Interface of the 14‐3‐3ζ Adapter Protein

14‐3‐3 Proteins play a central role in signalling pathways in cells: they interact as gatekeeper proteins with a huge number of binding partners. Their function as hub for intracellular communication can explain why these adapter proteins are associated with a wide range of diseases. How they control the various cellular mechanisms is still unclear, but it is assumed that the dimeric nature of the 14‐3‐3 proteins plays a key role in their activity. Here, we present, to the best of our knowledge, the first example of a small molecule binding to the 14‐3‐3ζ dimerisation interface. This compound was designed by rational in silico optimisation of a peptidic ligand identified from biochemical screening of a peptidic library, and the binding was characterised by UV/Vis spectroscopy, microscale thermophoresis, multiscale simulations, and X‐ray crystallography.     

Towards Functional Orthogonalisation of Protein Complexes: Individualisation of GroEL Monomers Leads to Distinct Quasihomogeneous Single Rings

The essential molecular chaperonin GroEL is an example of a functionally highly versatile cellular machine with a wide variety of in vitro applications ranging from protein folding to drug release. Directed evolution of new functions for GroEL is considered difficult, due to its structure as a complex homomultimeric double ring and the absence of obvious molecular engineering strategies. In order to investigate the potential to establish an orthogonal GroEL system in Escherichia coli, which might serve as a basis for GroEL evolution, we first successfully individualised groEL genes by inserting different functional peptide tags into a robustly permissive site identified by transposon mutagenesis. These peptides allowed fundamental aspects of the intracellular GroEL complex stoichiometry to be studied and revealed that GroEL single‐ring complexes, which assembled in the presence of several functionally equivalent but biochemically distinct monomers, each consist almost exclusively of only one type of monomer. At least in the case of GroEL, individualisation of monomers thus leads to individualisation of homomultimeric protein complexes, effectively providing the prerequisites for evolving an orthogonal intracellular GroEL folding machine.     

Structure and Function of Benzoylurea‐Derived α‐Helix Mimetics Targeting the Bcl‐xL/Bak Binding Interface

Targeting Bcl‐x _L /Bak : A family of rationally designed α‐helix mimetics with improved solubility and synthetic feasibility based on a benzoylurea scaffold is presented. These benzoylurea derivatives favor a linear conformation stabilized by an intramolecular hydrogen bond, and are able to mimic the spatial projection of the i, i+4, and i+7 residues of an α‐helix. Binding affinities of the benzoylurea derivatives to Bcl‐x_L have been assessed using fluorescence polarization competition assays and isothermal titration calorimetry.

     

A Pan‐Specific Inhibitor of the Polo‐Box Domains of Polo‐like Kinases Arrests Cancer Cells in Mitosis

Playing polo : Small‐molecule inhibitors of polo‐like kinase 1 are mostly ATP‐competitive, and thus face enormous specificity hurdles. This communication explores the concept of inhibiting Plk1 with a small‐molecule inhibitor of the protein–protein interactions required for Plk1 function.

     

Targeting a Dynamic Protein–Protein Interaction: Fragment Screening against the Malaria Myosin A Motor Complex

Motility is a vital feature of the complex life cycle of Plasmodium falciparum, the apicomplexan parasite that causes human malaria. Processes such as host cell invasion are thought to be powered by a conserved actomyosin motor (containing myosin A or myoA), correct localization of which is dependent on a tight interaction with myosin A tail domain interacting protein (MTIP) at the inner membrane of the parasite. Although disruption of this protein–protein interaction represents an attractive means to investigate the putative roles of myoA‐based motility and to inhibit the parasitic life cycle, no small molecules have been identified that bind to MTIP. Furthermore, it has not been possible to obtain a crystal structure of the free protein, which is highly dynamic and unstable in the absence of its natural myoA tail partner. Herein we report the de novo identification of the first molecules that bind to and stabilize MTIP via a fragment‐based, integrated biophysical approach and structural investigations to examine the binding modes of hit compounds. The challenges of targeting such a dynamic system with traditional fragment screening workflows are addressed throughout.