Robust Prediction of Relative Binding Energies for Protein-Protein Complex Mutations Using Free Energy Perturbation Calculations.

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations.

Biochemical Investigation of the Interaction of pICln, RioK1 and COPR5 with the PRMT5-MEP50 Complex.

The PRMT5-MEP50 methyltransferase complex plays a key role in various cancers and is regulated by different protein-protein interactions. Several proteins have been reported to act as adaptor proteins that recruit substrate proteins to the active site of PRMT5 for the methylation of arginine residues. To define the interaction between these adaptor proteins and PRMT5, we employed peptide truncation and mutation studies and prepared truncated protein constructs.

A protein tertiary structure mimetic modulator of the Hippo signalling pathway.

Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein-protein interactions (PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities.

Structure Based Design of Bicyclic Peptide Inhibitors of RbAp48.

The scaffolding protein RbAp48 is part of several epigenetic regulation complexes and is overexpressed in a variety of cancers. In order to develop tool compounds for the study of RbAp48 function, we have developed peptide inhibitors targeting the protein-protein interaction interface between RbAp48 and the scaffold protein MTA1. Based on a MTA1-derived linear peptide with low micromolar affinity and informed by crystallographic analysis, a bicyclic peptide was developed that inhibits the RbAp48/MTA1 interaction with a very low nanomolar K value of 8.

Discovery of Covalent Inhibitors Targeting the Transcriptional Enhanced Associate Domain Central Pocket.

Transcriptional enhanced associate domain (TEAD) transcription factors together with coactivators and corepressors modulate the expression of genes that regulate fundamental processes, such as organogenesis and cell growth, and elevated TEAD activity is associated with tumorigenesis. Hence, novel modulators of TEAD and methods for their identification are in high demand. We describe the development of a new "thiol conjugation assay" for identification of novel small molecules that bind to the TEAD central pocket.

TEAD-YAP Interaction Inhibitors and MDM2 Binders from DNA-Encoded Indole-Focused Ugi Peptidomimetics.

DNA-encoded combinatorial synthesis provides efficient and dense coverage of chemical space around privileged molecular structures. The indole side chain of tryptophan plays a prominent role in key, or "hot spot", regions of protein-protein interactions. A DNA-encoded combinatorial peptoid library was designed based on the Ugi four-component reaction by employing tryptophan-mimetic indole side chains to probe the surface of target proteins.

Lipidated Stapled Peptides Targeting the Acyl Binding Protein UNC119.

The acyl-binding UNC119 proteins mediate the activation and transport of various N-myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α-helical peptides that target UNC119a.

Molecular mechanism of polyketide shortening in anthraquinone biosynthesis of .

Anthraquinones, a widely distributed class of aromatic natural products, are produced by a type II polyketide synthase system in the Gram-negative bacterium . Heterologous expression of the anthraquinone biosynthetic gene cluster in identified AntI as an unusual lyase, catalysing terminal polyketide shortening prior to formation of the third aromatic ring. Functional and analysis of AntI using X-ray crystallography, structure-based mutagenesis, and molecular simulations revealed that AntI converts a defined octaketide to the tricyclic anthraquinone ring retro-Claisen and Dieckmann reactions.

Synthesis and SAR of the antistaphylococcal natural product nematophin from .

The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead structures are needed. Here, the synthesis and an expanded structure-activity relationship of the simple and antistaphylococcal amide nematophin from and synthetic derivatives are described.

Combined Approach of Backbone Amide Linking and On-Resin N-Methylation for the Synthesis of Bioactive and Metabolically Stable Peptides.

Rhabdopeptides are a large class of nonribosomal peptides from the bacteria Xenorhabdus and Photorhabdus with low micromolar activity against different protozoa, which are the causative agents of several tropical diseases. The development of a facile and flexible synthesis combining backbone amide linking with on-resin peralkylation for the synthesis of permethylated rhabdopeptides is described. This strategy allows the fast generation of permethylated naturally occurring and artificial rhabdopeptides for a structure-activity study.

New Modalities for Challenging Targets in Drug Discovery.

Our ever-increasing understanding of biological systems is providing a range of exciting novel biological targets, whose modulation may enable novel therapeutic options for many diseases. These targets include protein-protein and protein-nucleic acid interactions, which are, however, often refractory to classical small-molecule approaches. Other types of molecules, or modalities, are therefore required to address these targets, which has led several academic research groups and pharmaceutical companies to increasingly use the concept of so-called "new modalities".

Biosynthesis of the Antibiotic Nematophin and Its Elongated Derivatives in Entomopathogenic Bacteria.

Nematophin, a known antibiotic natural product against Staphylococcus aureus for almost 20 years, is produced by all strains of Xenorhabdus nematophila. Despite its simple structure, its biosynthesis was unknown. Its biosynthetic pathway is reported using heterologous production in Escherichia coli.

A straightforward method for automated Fmoc-based synthesis of bio-inspired peptide crypto-thioesters.

Despite recent advances, the direct Fmoc-based solid phase synthesis of peptide α-thioesters for the convergent synthesis of proteins native chemical ligation (NCL) remains a challenge in the field. We herein report a simple and general methodology, enabling access to peptide thioester surrogates. A novel C-terminal -(2-hydroxybenzyl)cysteine thioesterification device based on an amide-to-thioester rearrangement was developed, and the resulting peptide crypto-thioesters can be directly used in NCL reactions with fast → shift kinetics at neutral pH.

Solid-Phase Enrichment and Analysis of Azide-Labeled Natural Products: Fishing Downstream of Biochemical Pathways.

Many methods have been devised over the decades to trace precursors of specific molecules in cellular environments as, for example, in biosynthesis studies. The advent of click chemistry has facilitated the powerful combination of tracing and at the same time sieving the highly complex metabolome for compounds derived from simple or complex starting materials, especially when the click reaction takes place on a solid support. While the principle of solid-phase click reactions has already been successfully applied for selective protein and peptide enrichment, the successful enrichment of much smaller primary and secondary metabolites, showing great structural diversity and undergoing many different biosynthetic steps, has seen only little development.

Structure, Biosynthesis, and Occurrence of Bacterial Pyrrolizidine Alkaloids.

Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non-ribosomal peptide synthetase.

Selective fluorescent nonpeptidic antagonists for vasopressin V₂ GPCR: application to ligand screening and oligomerization assays.

A series of fluorescent benzazepine ligands for the arginine-vasopressin V₂ receptor (AVP V₂R) was synthesized using "Click" chemistry. Their in vitro pharmacological profile at AVP V₂R, V(1a)R, V(1b)R, and oxytocin receptor was measured by binding assay and functional studies. Compound 9p, labeled with Lissamine Rhodamine B using novel solid-phase organic tagging (SPOrT) resin, exhibited a high affinity for V₂R (4.