Automated Flow Peptide Synthesis Enables Engineering of Proteins with Stabilized Transient Binding Pockets.

Engineering at the amino acid level is key to enhancing the properties of existing proteins in a desired manner. So far, protein engineering has been dominated by genetic approaches, which have been extremely powerful but only allow for minimal variations beyond the canonical amino acids. Chemical peptide synthesis allows the unrestricted incorporation of a vast set of unnatural amino acids with much broader functionalities, including the incorporation of post-translational modifications or labels. Here we demonstrate the potential of chemical synthesis to generate proteins in a specific conformation, which would have been unattainable by recombinant protein expression. We use recently established rapid automated flow peptide synthesis combined with solid-phase late-stage modifications to rapidly generate a set of FK506-binding protein 51 constructs bearing defined intramolecular lactam bridges. This trapped an otherwise rarely populated transient pocket-as confirmed by crystal structures-which led to an up to 39-fold improved binding affinity for conformation-selective ligands and represents a unique system for the development of ligands for this rare conformation. Overall, our results show how rapid automated flow peptide synthesis can be applied to precision protein engineering.

Synthetic Peptides: Promising Modalities for the Targeting of Disease-Related Nucleic Acids.

DNA and RNA play pivotal roles in life processes by storing and transferring genetic information, modulating gene expression, and contributing to essential cellular machinery such as ribosomes. Dysregulation and mutations in nucleic acid-related processes are implicated in numerous diseases. Despite the critical impact on health of nucleic acid mutations or dysregulation, therapeutic compounds addressing these biomolecules remain limited. Peptides have emerged as a promising class of molecules for biomedical research, offering potential solutions for challenging drug targets. This review focuses on the use of synthetic peptides to target disease-related nucleic acids. We discuss examples of peptides targeting double-stranded DNA, including the clinical candidate Omomyc, and compounds designed for regulatory G-quadruplexes. Further, we provide insights into both library-based screenings and the rational design of peptides to target regulatory human RNA scaffolds and viral RNAs, emphasizing the potential of peptides in addressing nucleic acid-related diseases.

Use of DNA forceps to measure receptor-ligand dissociation equilibrium constants in a single-molecule competition assay.

The ability of biophysicists to decipher the behavior of individual biomolecules has steadily improved over the past thirty years. However, it still remains unclear how an ensemble of data acquired at the single-molecule level compares with the data acquired on an ensemble of the same molecules. We here propose an assay to tackle this question in the context of dissociation equilibrium constant measurements. A sensor is built by engrafting a receptor and a ligand onto a flexible dsDNA scaffold and mounting this assembly on magnetic tweezers. This way, looking at the position of the magnetic bead enables one to determine in real-time if the two molecular partners are associated or not. Next, to quantify the affinity of the scrutinized single-receptor for a given competitor, various amounts of the latter molecule are introduced in solution and the equilibrium response of the sensor is monitored throughout the titration protocol. Proofs of concept are established for the binding of three rapamycin analogs to the FKBP12 cis-trans prolyl isomerase. For each of these drugs the mean affinity constant obtained on a ten of individual receptors agrees with the one previously determined in a bulk assay. Furthermore, experimental contingencies are sufficient to explain the dispersion observed over the single-molecule values.

Advances in Ultrahigh Throughput Hit Discovery with Tandem Mass Spectrometry Encoded Libraries.

Discovering new bioactive molecules is crucial for drug development. Finding a hit compound for a new drug target usually requires screening of millions of molecules. Affinity selection based technologies have revolutionized early hit discovery by enabling the rapid screening of libraries with millions or billions of compounds in short timeframes. In this Perspective, we describe recent technology breakthroughs that enable the screening of ultralarge synthetic peptidomimetic libraries with a barcode-free tandem mass spectrometry decoding strategy. A combination of combinatorial synthesis, affinity selection, automated de novo peptide sequencing algorithms, and advances in mass spectrometry instrumentation now enables hit discovery from synthetic libraries with over 100 million members. We provide a perspective on this powerful technology and showcase success stories featuring the discovery of high affinity binders for a number of drug targets including proteins, nucleic acids, and specific cell types. Further, we show the usage of the technology to discover synthetic peptidomimetics with specific functions and reactivity. We predict that affinity selection coupled with tandem mass spectrometry and automated de novo decoding will rapidly evolve further and become a broadly used drug discovery technology.

Picomolar FKBP inhibitors enabled by a single water-displacing methyl group in bicyclic [4.3.1] aza-amides.

Methyl groups can have profound effects in drug discovery but the underlying mechanisms are diverse and incompletely understood. Here we report the stereospecific effect of a single, solvent-exposed methyl group in bicyclic [4.3.1] aza-amides, robustly leading to a 2 to 10-fold increase in binding affinity for FK506-binding proteins (FKBPs). This resulted in the most potent and efficient FKBP ligands known to date. By a combination of co-crystal structures, isothermal titration calorimetry (ITC), density-functional theory (DFT), and 3D reference interaction site model (3D-RISM) calculations we elucidated the origin of the observed affinity boost, which was purely entropically driven and relied on the displacement of a water molecule at the protein-ligand-bulk solvent interface. The best compounds potently occupied FKBPs in cells and enhanced bone morphogenic protein (BMP) signaling. Our results show how subtle manipulation of the solvent network can be used to design atom-efficient ligands for difficult, solvent-exposed binding pockets.

Parallel Automated Flow Synthesis of Covalent Protein Complexes That Can Inhibit MYC-Driven Transcription.

Dysregulation of the transcription factor MYC is involved in many human cancers. The dimeric transcription factor complexes of MYC/MAX and MAX/MAX activate or inhibit, respectively, gene transcription upon binding to the same enhancer box DNA. Targeting these complexes in cancer is a long-standing challenge. Inspired by the inhibitory activity of the MAX/MAX dimer, we engineered covalently linked, synthetic homo- and heterodimeric protein complexes to attenuate oncogenic MYC-driven transcription. We prepared the covalent protein complexes (∼20 kDa, 167-231 residues) in a single shot via parallel automated flow synthesis in hours. The stabilized covalent dimers display DNA binding activity, are intrinsically cell-penetrant, and inhibit cancer cell proliferation in different cell lines. RNA sequencing and gene set enrichment analysis in A549 cancer cells confirmed that the synthetic dimers interfere with MYC-driven transcription. Our results demonstrate the potential of automated flow technology to rapidly deliver engineered synthetic protein complex mimetics that can serve as a starting point in developing inhibitors of MYC-driven cancer cell growth.

Engineering Bioactive Dimeric Transcription Factor Analogs via Palladium Rebound Reagents.

Transcription factors (TF), such as Myc, are proteins implicated in disease pathogenesis, with dysregulation of Myc expression in 50% of all human cancers. Still, targeting Myc remains a challenge due to the lack of small molecule binding pockets in the tertiary structure. Here, we report synthetic covalently linked TF mimetics that inhibit oncogenic Myc-driven transcription by antagonistic binding of the target DNA-binding site. We combined automated flow peptide chemistry with palladium(II) oxidative addition complexes (OACs) to engineer covalent protein dimers derived from the DNA-binding domains of Myc, Max, and Omomyc TF analogs. Palladium-mediated cross-coupling of synthesized protein monomers resulted in milligram quantities of seven different covalent homo- and heterodimers. The covalent helical dimers were found to bind DNA and exhibited improved thermal stability. Cell-based studies revealed the Max-Max covalent dimer is cell-penetrating and interfered with Myc-dependent gene transcription resulting in reduced cancer cell proliferation (EC50 of 6 µM in HeLa). RNA sequencing and gene analysis of extracted RNA from treated cancer cells confirmed that the covalent Max-Max homodimer interferes with Myc-dependent transcription. Flow chemistry, combined with palladium(II) OACs, has enabled a practical strategy to generate new bioactive compounds to inhibit tumor cell proliferation.

De Novo Discovery of High-Affinity Peptide Binders for the SARS-CoV-2 Spike Protein.

The ß-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein are of interest for the development of therapeutics and diagnostics. We used affinity selection-mass spectrometry for the rapid discovery of synthetic high-affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. From library screening with 800 million synthetic peptides, we identified three sequences with nanomolar affinities (dissociation constants K d = 80-970 nM) for RBD and selectivity over human serum proteins. Nanomolar RBD concentrations in a biological matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides do not compete for ACE2 binding, and their site of interaction on the SARS-CoV-2-spike-RBD might be unrelated to the ACE2 binding site, making them potential orthogonal reagents for sandwich immunoassays. These findings serve as a starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus-directed delivery of therapeutics.

Discovery of Nucleic Acid Binding Molecules from Combinatorial Biohybrid Nucleobase Peptide Libraries.

Nature has three biopolymers: oligonucleotides, polypeptides, and oligosaccharides. Each biopolymer has independent functions, but when needed, they form mixed assemblies for higher-order purposes, as in the case of ribosomal protein synthesis. Rather than forming large complexes to coordinate the role of different biopolymers, we dovetail protein amino acids and nucleobases into a single low molecular weight precision polyamide polymer. We established efficient chemical synthesis and de novo sequencing procedures and prepared combinatorial libraries with up to 100 million biohybrid molecules. This biohybrid material has a higher bulk affinity to oligonucleotides than peptides composed exclusively of canonical amino acids. Using affinity selection mass spectrometry, we discovered variants with a high affinity for pre-microRNA hairpins. Our platform points toward the development of high throughput discovery of sequence defined polymers with designer properties, such as oligonucleotide binding.

Secondary Amino Alcohols: Traceless Cleavable Linkers for Use in Affinity Capture and Release.

Capture and release of peptides is often a critical operation in the pathway to discovering materials with novel functions. However, the best methods for efficient capture impede facile release. To overcome this challenge, we report linkers based on secondary amino alcohols for the release of peptides after capture. These amino alcohols are based on serine (seramox) or isoserine (isoseramox) and can be incorporated into peptides during solid-phase peptide synthesis through reductive amination. Both linkers are quantitatively cleaved within minutes under NaIO4 treatment. Cleavage of isoseramox produced a native peptide N-terminus. This linker also showed broad substrate compatibility; incorporation into a synthetic peptide library resulted in the identification of all sequences by nanoLC-MS/MS. The linkers are cell compatible; a cell-penetrating peptide that contained this linker was efficiently captured and identified after uptake into cells. These findings suggest that such secondary amino alcohol based linkers might be suitable tools for peptide-discovery platforms.

Targeting the SARS-CoV-2-spike protein: from antibodies to miniproteins and peptides.

Coronavirus disease-19, caused by the novel ß-coronavirus SARS-CoV-2, has created a global pandemic unseen in a century. Rapid worldwide efforts have enabled the characterization of the virus and its pathogenic mechanism. An early key finding is that SARS-CoV-2 uses spike proteins, the virus' most exposed structures, to bind to human ACE2 receptors and initiate cell invasion. Competitive targeting of the spike protein is a promising strategy to neutralize virus infectivity. This review article summarizes the discovery, binding modes and eventual applications of several classes of (bio)molecules targeting the spike protein: antibodies, nanobodies, soluble ACE2 variants, miniproteins, peptides and small molecules.

Efficient Pictet-Spengler Bioconjugation with N-Substituted Pyrrolyl Alanine Derivatives.

We discovered N-pyrrolyl alanine derivatives as efficient reagents for the fast and selective Pictet-Spengler reaction with aldehyde-containing biomolecules. Other aldehyde-labeling methods described so far have several drawbacks, like hydrolytic instability, slow reaction kinetics or not readily available labeling reagents. Pictet-Spengler cyclizations of pyrrolyl 2-ethylamine substituted at the pyrrole nitrogen are significantly faster than with analogues substituted at the α- and ß- position. Functionalized N-pyrrolyl alanine derivatives can be synthesized in only 2-3 steps from commercially available materials. The small size of the reagent, the high reaction rate, and the easy synthesis make pyrrolyl alanine Pictet-Spengler (PAPS) an attractive choice for bioconjugation reactions. PAPS was shown as an efficient strategy for the site-selective biotinylation of an antibody as well as for the condensation of nucleic-acid derivatives, demonstrating the versatility of this reagent.

Chemogenomic Profiling of Human and Microbial FK506-Binding Proteins.

FK506-binding proteins (FKBPs) are evolutionarily conserved proteins that display peptidyl-prolyl isomerase activities and act as coreceptors for immunosuppressants. Microbial macrophage-infectivity-potentiator (Mip)-type FKBPs can enhance infectivity. However, developing druglike ligands for FKBPs or Mips has proven difficult, and many FKBPs and Mips still lack biologically useful ligands. To explore the scope and potential of C5-substituted [4.3.1]-aza-bicyclic sulfonamides as a broadly applicable class of FKBP inhibitors, we developed a new synthesis method for the bicyclic core scaffold and used it to prepare an FKBP- and Mip-focused library. This allowed us to perform a systematic structure-activity-relationship analysis across key human FKBPs and microbial Mips, yielding highly improved inhibitors for all the FKBPs studied. A cocrystal structure confirmed the molecular-binding mode of the core structure and explained the affinity gained as a result of the preferred substituents. The best FKBP and Mip ligands showed promising antimalarial, antileginonellal, and antichlamydial properties in cellular models of infectivity, suggesting that substituted [4.3.1]-aza-bicyclic sulfonamides could be a novel class of anti-infectives.

Recent Progress in FKBP Ligand Development.

FK506-binding proteins have been implicated in numerous human diseases suggesting novel therapeutic opportunities. In particular, the large FKBP51 has emerged as an important regulator of the stress-coping system and as an established risk factor for stress-related disorders. The principal druggabilty of FKBPs is evidenced by the prototypical high affinity ligands FK506 and rapamycin but the development of more refined and selective chemical probes for FKBPs has been challenging. In this review we summarize recent advances in the development of FKBP ligands, which cumulated in the first highly selective ligands for FKBP51. The best ligand SAFit2 allowed the proof-of-concept in mice for FKBP51 inhibitors as potentially novel antidepressants. Finally, we discuss pending issues that need to be addressed for the further development of FKBP51-directed drugs.

Rational design and asymmetric synthesis of potent and neurotrophic ligands for FK506-binding proteins (FKBPs).

To create highly efficient inhibitors for FK506-binding proteins, a new asymmetric synthesis for pro-(S)-C(5) -branched [4.3.1] aza-amide bicycles was developed. The key step of the synthesis is an HF-driven N-acyliminium cyclization. Functionalization of the C(5)  moiety resulted in novel protein contacts with the psychiatric risk factor FKBP51, which led to a more than 280-fold enhancement in affinity. The most potent ligands facilitated the differentiation of N2a neuroblastoma cells with low nanomolar potency.

Loratadine and analogues: discovery and preliminary structure-activity relationship of inhibitors of the amino acid transporter B(0)AT2.

B(0)AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain, but little is otherwise known about its function. To identify inhibitors for this transporter, we screened a library of 3133 different bioactive compounds. Loratadine, a clinically used histamine H1 receptor antagonist, was identified as a selective inhibitor of B(0)AT2 with an IC50 of 4 µM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B(0)AT2 was confirmed by electrophysiology. A series of loratadine analogues were synthesized to gain insight into the structure-activity relationships. Our studies provide the first chemical tool for B(0)AT2.