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. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.

The European Federation for Medicinal Chemistry and Chemical Biology (EFMC) Best Practice Initiative: Hit Generation.

Hit generation is a crucial step in drug discovery that will determine the speed and chance of success of identifying drug candidates. Many strategies are now available to identify chemical starting points, or hits, and each biological target warrants a tailored approach. In this set of best practices, we detail the essential approaches for target centric hit generation and the opportunities and challenges they come with. We then provide guidance on how to validate hits to ensure medicinal chemistry is only performed on compounds and scaffolds that engage the target of interest and have the desired mode of action. Finally, we discuss the design of integrated hit generation strategies that combine several approaches to maximize the chance of identifying high quality starting points to ensure a successful drug discovery campaign.

The European Federation for Medicinal Chemistry and Chemical Biology (EFMC) Best Practice Initiative: Phenotypic Drug Discovery.

Phenotypic drug discovery has a long track record of delivering innovative drugs and has received renewed attention in the last few years. The promise of this approach, however, comes with several challenges that should be addressed to avoid wasting time and resources on drugs with undesired modes of action or, worse, false-positive hits. In this set of best practices, we go over the essential steps of phenotypic drug discovery and provide guidance on how to increase the chance of success in identifying validated and relevant chemical starting points for optimization: selecting the right assay, selecting the right compound screening library and developing appropriate hit validation assays. Then, we highlight the importance of initiating studies to determine the mode of action of the identified hits early and present the current state of the art.

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. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators.

The European Federation for Medicinal Chemistry (EFMC) Best Practice Initiative: Validating Chemical Probes.

As part of an initiative aimed to share best practices in Medicinal Chemistry, the European Federation for Medicinal Chemistry (EFMC) is preparing a series of webinars and slide sets focused on the early phase of drug discovery. This educational material is freely accessible through the EFMC. The main target audiences are students or early career scientists and we also believe it will be valuable for experienced practitioners. The first of the series is focused on the generation and validation of high-quality chemical probes, which are critical for drug discovery and more broadly to further our understanding of human biology and disease.

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. The assay monitors prevention of covalent binding of a fluorescence turn-on probe to a cysteine in the central pocket by small molecules. Screening of a collection of compounds revealed kojic acid analogues as TEAD inhibitors, which covalently target the cysteine in the central pocket, block the interaction with coactivator yes-associated protein with nanomolar apparent IC50 values, and reduce TEAD target gene expression. This methodology promises to enable new medicinal chemistry programs aimed at the modulation of TEAD activity.

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. Several peptoids were synthesized on a chemically stable hexathymidine adapter oligonucleotide "hexT", encoded by DNA sequences, and substituted by azide-alkyne cycloaddition to yield a library of 8112 molecules. Selection experiments for the tumor-relevant proteins MDM2 and TEAD4 yielded MDM2 binders and a novel class of TEAD-YAP interaction inhibitors that perturbed the expression of a gene under the control of these Hippo pathway effectors.

Macrocyclic Modalities Combining Peptide Epitopes and Natural Product Fragments.

"Hot loop" protein segments have variable structure and conformation and contribute crucially to protein-protein interactions. We describe a new hot loop mimicking modality, termed PepNats, in which natural product (NP)-inspired structures are incorporated as conformation-determining and -restricting structural elements into macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were synthesized on solid support employing macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors. NP-inspired fragment absolute configuration determines the conformation of the peptide part responsible for binding. These results demonstrate that combination of NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop mimics with conformationally constrained and biologically relevant structure.

Identification of Quinolinols as Activators of TEAD-Dependent Transcription.

The transcriptional co-regulators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are the vertebrate downstream effectors of the Hippo signaling pathway that controls various physiological and pathological processes. YAP and TAZ pair with the TEAD (TEA domain) family of transcription factors to initiate transcription. We previously identified a tractable pocket in TEADs, which has been physiologically shown to bind palmitate. Herein, a TEAD-palmitate interaction screen was developed to select small molecules occupying the palmitate-binding pocket (PBP) of TEADs. We show that quinolinols were TEAD-binding compounds that augment YAP/TAZ-TEAD activity, which was verified using TEAD reporter assay, RT-qPCR, and RNA-Seq analyses. Structure-activity relationship investigations uncovered the quinolinol substituents that are necessary for TEAD activation. We reveal a novel mechanism where quinolinols stabilize YAP/TAZ protein levels by occupying the PBP. The enhancement of YAP activity by quinolinols accelerates the in vivo wound closure in a mouse wound-healing model. Although small molecules that occupy the PBP have been shown to inhibit YAP/TAZ-TEAD activity, leveraging PBP to activate TEADs is a novel approach.

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". This Review defines for the first time the scope of this term, which includes novel peptidic scaffolds, oligonucleotides, hybrids, molecular conjugates, as well as new uses of classical small molecules. We provide the most representative examples of these modalities to target large binding surface areas such as those found in protein-protein interactions and for biological processes at the center of cell regulation.

A cyclic carbo-isosteric penta-depsipeptide: cyclo(Phe(1)-d-Ala(2)-Gly(3)-Phe(4)-APO(5)).

The title compound, cyclo(Phe(1)-d-Ala(2)-Gly(3)-Phe(4)-APO(5)), C26H32N4O5, is the minor diastereoisomer of a cyclic penta-peptidomimetic analogue containing a novel 2-amino-propyl lactone (APO) motif, which displays the same number of atoms as the native amino acid glycine and has a methyl group in place of the carbonyl O atom. The crystal structure presented here allows the analysis of the secondary structure of this unprecedented cyclic carbo-isosteric depsipeptide. The conformation of the central ring is stabilized by an intra-molecular N-H⋯O hydrogen bond between the carbonyl O atom of the first residue (Phe(1)) and the amide group H atom of the fourth residue (Phe(4)). Based on the previously reported hydrogen bond and on the values of the torsion angles ϕ and ψ, the loop formed by the first, second, third and fourth residues (Phe(1), d-Ala(2), Gly(3) and Phe(4)) can be classified as a type II' ß-turn. The loop around the new peptidomimetic motif, on the other hand, resembles an open γ-turn containing a weak N-H⋯O hydrogen bond between the carbonyl group O atom of the fourth residue (Phe(4)) and the amide unit H atom of the first residue (Phe(1)). In the crystal, the peptidomimetic mol-ecules are arranged in chains along the b-axis direction. Within such a chain, the mol-ecules of the structure are linked via N-H⋯O hydrogen bonds between the amide group H atom of the secondary residue (d-Ala(2)) and the carb-oxy unit O atom of the fourth residue (Phe(4)) in a neighboring mol-ecule. The newly formed methyl stereocentre of the APO peptidomimetic motif (APO(5)) was obtained as the minor diastereoisomer in a ring-closing reductive amination reaction and adopts an R configuration.

Cyclic carbo-isosteric depsipeptides and peptides as a novel class of peptidomimetics.

A novel and highly efficient cyclization method has been developed to access a new class of cyclic carbo-isosteric depsipeptides and carbo-isosteric peptides. Our strategy requires easily accessible C-terminal methyl ketone ester or amide functionalized linear precursors as starting materials. The well-known reductive amination has then been used to afford cyclic tetra- to octa-pseudopeptides via a selective intramolecular formation of a glycine peptidomimetic unit under moderate dilution.

Synthesis of a functionalized 7,6-bicyclic spiroimine ring fragment of the spirolides.

The asymmetric synthesis of a functionalized 7,6-spiroimine related to the spirolides is described. Intermolecular Diels-Alder cycloaddition of a chiral trisubstituted dienophile and Danishefsky's diene enabled simultaneous installation of the C7 and C29 stereocenters. Further transformations and late-stage aza-Wittig cyclization afforded the spiroimine in good yield. During this study, an unprecedented 14-membered dialdimine was also obtained.

(1R,6R,13R,18R)-(Z,Z)-1,18-Bis[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]-3,16-dimethyl-ene-8,20-diaza-dispiro-[5.6.5.6]tetra-cosa-7,19-diene.

The crystal structure of the title compound, C(34)H(54)N(2)O(4), has been solved in order to prove the relative and absolute chirality of the newly-formed stereocentres which were established using an asymmetric Diels-Alder reaction at an earlier stage in the synthesis. This unprecedented stable dialdimine contains a 14-membered ring and was obtained as the minor diastereoisomer in the Diels-Alder reaction. The absolute stereochemistry of the stereocentres of the acetal functionality was known to be R based on the use of a chiral (R)-tris-ubstituted dienophile derived from enanti-opure (S)-glyceraldehyde. The assignment of the configuration in the dienophile and the title di-aldimine differs from (S)-glyceraldehyde due to a change in the priority order of the substituents. The crystal structure establishes the presence of six stereocentres all attributed to be R. The 14-membered ring contains two aldimine bonds [C-N = 1.258 (2) and 1.259 (2) Å]. It adopts a similar conformation to that proposed for trans-trans-cyclo-tetra-deca-1,8-dienes.

Synthesis and reactivity of beta-methoxymethyl enecarbamates.

Beta-methoxymethyl enecarbamates (e.g., 1) have been prepared in a single step from alpha-methoxy carbamates. In the presence of a mild Lewis acid, compound 1 underwent substitution with a variety of nucleophiles including indoles, electron-rich aromatics, silyl enol ethers, and 2-trimethylsilyloxyfuran.

Synthesis of enantiopure bicyclic alpha,alpha-disubstituted spirolactams via asymmetric Birch reductive alkylation.

The synthesis of enantiopure bicyclic alpha,alpha-disubstituted spirolactams is described using a diastereoselective Birch reductive alkylation as the key step. Hydrogenation of the resultant alkylated cyclohexadienes followed by intramolecular cyclization provides access to enantiopure 8-azaspiro[5.6]dodecan-7-ones.

(1R,6R)-1-Methyl-8-aza-spiro-[5.6]dodecan-7-one.

The crystal structure of the title compound, C(12)H(21)NO, has been investigated to establish the absolute stereochemistry at position 1. The absolute stereochemistry at the quaternary centre at position 6 is established to be R using an asymmetric Birch reductive alkyl-ation reaction for which the stereochemical outcome is known. The crystal structure indicates the presence of two conformers of the bicyclic (1R,6R)-spiro-lactam ring system that differ in the conformation adopted by the six-membered ring. In one conformer, the meth-yl group adopts an axial position whereas in the other conformer, the same methyl group adopts an equatorial position. In both conformers, the seven-membered ring adopts a chair conformation. The two conformers of the bicyclic spiro-lactam are connected to each other via inter-molecular N-H⋯O hydrogen bonds forming a heterodimer. The asymmetric unit contains two such dimers.