Blocking of the PD‐1/PD‐L1 Interaction by a D‐Peptide Antagonist for Cancer Immunotherapy

Blockade of the protein–protein interaction between the transmembrane protein programmed cell death protein 1 (PD‐1) and its ligand PD‐L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror‐image phage display, we developed the first hydrolysis‐resistant D ‐peptide antagonists to target the PD‐1/PD‐L1 pathway. The optimized compound ^DPPA‐1 could bind PD‐L1 at an affinity of 0.51 μM in vitro. A blockade assay at the cellular level and tumor‐bearing mice experiments indicated that ^DPPA‐1 could also effectively disrupt the PD‐1/PD‐L1 interaction in vivo. Thus D ‐peptide antagonists may provide novel low‐molecular‐weight drug candidates for cancer immunotherapy.     

A 3.3 Å‐Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability

Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc₁ complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc₁ complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c₁ was found to greatly enhance the interaction between cyt. b and cyt. c₁, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc₁ complex in an extreme thermal environment.     

A Modular Synthesis of Teraryl‐Based α‐Helix Mimetics,Part 1: Synthesis of Core Fragments with Two Electronically Differentiated Leaving Groups

Teraryl‐based α‐helix mimetics have proven to be useful compounds for the inhibition of protein–protein interactions (PPI). We have developed a modular and flexible approach for the synthesis of teraryl‐based α‐helix mimetics. Central to our strategy is the use of a benzene core unit featuring two leaving groups of differentiated reactivity in the Pd‐catalyzed cross‐coupling used for terphenyl assembly. With the halogen/diazonium route and the halogen/triflate route, two strategies have successfully been established. The synthesis of core building blocks with aliphatic (Ala, Val, Leu, Ile), aromatic (Phe), polar (Cys, Lys), hydrophilic (Ser, Gln), and acidic (Glu) amino acid side chains are reported.     

Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC‐associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA‐based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC‐associated proteins. This study provided a simple and broadly applicable method for characterizing protein‐protein interactions.     

A Novel d‐Peptide Identified by Mirror‐Image Phage Display Blocks TIGIT/PVR for Cancer Immunotherapy

The low response rate and adaptive resistance of PD‐1/PD‐L1 blockade demands the studies on novel therapeutic targets for cancer immunotherapy. We discovered that a novel immune checkpoint TIGIT expressed higher than PD‐1 in many tumors especially anti‐PD‐1 resistant tumors. Here, mirror‐image phage display bio‐panning was performed using the d ‐enantiomer of TIGIT synthesized by hydrazide‐based native chemical ligation. d ‐peptide ^DTBP‐3 was identified, which could occupy the binding interface and effectively block the interaction of TIGIT with its ligand PVR. ^DTBP‐3 showed proteolytic resistance, tumor tissue penetrating ability, and significant tumor suppressing effects in a CD8⁺ T cell dependent manner. More importantly, ^DTBP‐3 could inhibit tumor growth and metastasis in anti‐PD‐1 resistant tumor model. This is the first d ‐peptide targeting TIGIT, which could serve as a potential candidate for cancer immunotherapy.     

Accelerating the Association of the Most Stable Protein–Ligand Complex by More than Two Orders of Magnitude

The complex between the bacterial type 1 pilus subunit FimG and the peptide corresponding to the N‐terminal extension (termed donor strand, Ds) of the partner subunit FimF (DsF) shows the strongest reported noncovalent molecular interaction, with a dissociation constant (K_D) of 1.5×10^?20 m . However, the complex only exhibits a slow association rate of 330 m ^?1 s^?1 that limits technical applications, such as its use in affinity purification. Herein, a structure‐based approach was used to design pairs of FimGt (a FimG variant lacking its own N‐terminal extension) and DsF variants with enhanced electrostatic surface complementarity. Association of the best mutant FimGt/DsF pairs was accelerated by more than two orders of magnitude, while the dissociation rates and 3D structures of the improved complexes remained essentially unperturbed. A K_D value of 8.8×10^?22 m was obtained for the best mutant complex, which is the lowest value reported to date for a protein/ligand complex.     

A Small‐Molecule Protein–Protein Interaction Inhibitor of PARP1 That Targets Its BRCT Domain

Poly(ADP‐ribose)polymerase‐1 (PARP1) is a BRCT‐containing enzyme (BRCT=BRCA1 C‐terminus) mainly involved in DNA repair and damage response and a validated target for cancer treatment. Small‐molecule inhibitors that target the PARP1 catalytic domain have been actively pursued as anticancer drugs, but are potentially problematic owing to a lack of selectivity. Compounds that are capable of disrupting protein–protein interactions of PARP1 provide an alternative by inhibiting its activities with improved selectivity profiles. Herein, by establishing a high‐throughput microplate‐based assay suitable for screening potential PPI inhibitors of the PARP1 BRCT domain, we have discovered that (±)‐gossypol, a natural product with a number of known biological activities, possesses novel PARP1 inhibitory activity both in vitro and in cancer cells and presumably acts through disruption of protein–protein interactions. As the first known cell‐permeable small‐molecule PPI inhibitor of PAPR1, we further established that (?)‐gossypol was likely the causative agent of PARP1 inhibition by promoting the formation of a 1:2 compound/PARP1 complex by reversible formation of a covalent imine linkage.     

A Modular Synthesis of Teraryl‐Based α‐Helix Mimetics,Part 2: Synthesis of 5‐Pyridine Boronic Acid Pinacol Ester Building Blocks with Amino Acid Side Chains in 3‐Position

One of the most common protein–protein interactions (PPI) is the interaction of the α‐helix of one protein with the surface of the second one. Terphenylic scaffolds are bioinspired motifs in the inhibition of PPIs and have been identified as suitable α‐helix mimetics. One of the challenging aspects of this strategy is the poor solubility of terphenyls under physiological conditions. In the literature pyrrolopyrimidine‐, pyrimidine‐ or pyridazine‐based mimetics have been reported to show improved solubility. We present a new convergent strategy for the synthesis of linear pyridine‐type teraryls based on a phenylic core unit. A general approach for the synthesis of 3,5‐disubstituted pyridine‐based boronic acid pinacol esters with amino acid side chains in the 3‐position (representing Phe, Leu, Ile, Lys, Asp, Asn) is presented and exploits the functional group tolerance of the Knochel–Grignard reagents. The building blocks have been used in a convergent in situ two‐step synthesis of teraryl α‐helix mimetics.     

Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen‐binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single‐domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o‐Nitrobenzyl‐protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti‐GFP photobody exhibited an at least 10 000‐fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody–photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.     

Contact between the β1 and β2 Segments of α‐Synuclein that Inhibits Amyloid Formation

Conversion of the intrinsically disordered protein α‐synuclein (α‐syn) into amyloid aggregates is a key process in Parkinson’s disease. The sequence region 35–59 contains β‐strand segments β1 and β2 of α‐syn amyloid fibril models and most disease‐related mutations. β1 and β2 frequently engage in transient interactions in monomeric α‐syn. The consequences of β1–β2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double‐cysteine mutant α‐synCC, with a disulfide linking β1 and β2, is aggregation‐incompetent and inhibits aggregation and toxicity of wild‐type α‐syn. We show that α‐syn delays the aggregation of amyloid‐β peptide and islet amyloid polypeptide involved in Alzheimer’s disease and type 2 diabetes, an effect enhanced in the α‐synCC mutant. Tertiary interactions in the β1–β2 region of α‐syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach.     

Oxime Side‐Chain Cross‐Links in an α‐Helical Coiled‐Coil Protein: Structure,Thermodynamics, and Folding‐Templated Synthesis of Bicyclic Species

Covalent side‐chain cross‐links are a versatile method to control peptide folding, particularly when α‐helical secondary structure is the target. Here, we examine the application of oxime bridges, formed by the chemoselective reaction between aminooxy and aldehyde side chains, for the stabilization of a helical peptide involved in a protein–protein complex. A series of sequence variants of the dimeric coiled coil GCN4‐p1 bearing oxime bridges at solvent‐exposed positions were prepared and biophysically characterized. Triggered unmasking of a side‐chain aldehyde in situ and subsequent cyclization proceed rapidly and cleanly at pH 7 in the folded protein complex. Comparison of folding thermodynamics among a series of different oxime bridges show that the cross links are consistently stabilizing to the coiled coil, with the extent of stabilization sensitive to the exact size and structure of the macrocycle. X‐ray crystallographic analysis of a coiled coil with the best cross link in place and a second structure of its linear precursor show how the bridge is accommodated into an α‐helix. Preparation of a bicyclic oligomer by simultaneous formation of two linkages in situ demonstrates the potential use of triggered oxime formation to both trap and stabilize a particular peptide folded conformation in the bound state.     

Free‐Standing Gold‐Nanoparticle Monolayer Film Fabricated by Protein Self‐Assembly of α‐Synuclein

Free‐standing nanoparticle films are of great importance for developing future nano‐electronic devices. We introduce a protein‐based fabrication strategy of free‐standing nanoparticle monolayer films. α‐Synuclein, an amyloidogenic protein, was utilized to yield a tightly packed gold‐nanoparticle monolayer film interconnected by protein β‐sheet interactions. Owing to the stable protein–protein interaction, the film was successfully expanded to a 4‐inch diameter sheet, which has not been achieved with any other free‐standing nanoparticle monolayers. The film was flexible in solution, so it formed a conformal contact, surrounding even microspheres. Additionally, the monolayer film was readily patterned at micrometer‐scale and thus unprecedented double‐component nanoparticle films were fabricated. Therefore, the free‐floating gold‐nanoparticle monolayer sheets with these properties could make the film useful for the development of bio‐integrated nano‐devices and high‐performance sensors.     

Unconventional Secondary Structure Mimics: Ladder‐Rungs

Secondary structures tend to be recognizable because they have repeating structural motifs, but mimicry of these does not have to follow such well‐defined patterns. Bioinformatics studies to match side‐chain orientations of a novel hydantoin triazole chemotype ( 1 ) to protein‐protein interfaces revealed it tends to align well across parallel and antiparallel sheets, like rungs on a ladder. One set of these overlays was observed for the protein‐protein interaction uPA?uPAR. Consequently, chemotype 1 was made with appropriate side‐chains to mimic uPA at this interface. Biophysical assays indicate these compounds did in fact bind uPAR, and elicit cellular responses that affected invasion, migration, and wound healing.     

High‐Resolution Structural Characterization of a Helical α/β‐Peptide Foldamer Bound to the Anti‐Apoptotic Protein Bcl‐xL

Get into the groove : The first high‐resolution structure of a foldamer bound to a protein target is described (see picture; foldamer in sticks). The foldamer consists of α‐ and β‐amino acid residues and is bound to the anti‐apoptotic protein Bcl‐x_L. The overall binding mode and key interactions observed in the foldamer/Bcl‐x_L complex mimic those seen in complexes of Bcl‐x_L with natural α‐peptide ligands. Additional contacts in the foldamer/Bcl‐x_L complex involving β‐amino acid residues appear to contribute to binding affinity.