Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein-Protein Interaction from Nonselective Fragments.

The stabilization of protein-protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3σ bound to several of its clients, including ERα and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3σ/ERα complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3σ/C-RAF up to 150 µM compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure-activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3σ/ERα interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development.

Pd-Catalyzed de Novo Assembly of Diversely Substituted Indole-Fused Polyheterocycles.

Here we describe a facile, tandem synthetic route for indolo[3,2-c]quinolinones, a class of natural alkaloid analogues of high biological significance. A Ugi four-component reaction with indole-2-carboxylic acid and an aniline followed by a Pd-catalyzed cyclization yields tetracyclic indoloquinolines in good to moderate yields. Commercially available building blocks yield highly diverse analogues in just two simple steps.

ß-Carbolinone Analogues from the Ugi Silver Mine.

Here we describe a facile, tandem synthetic route for ß-carbolinones, a class of natural products of high biological significance. Commercially available building blocks yield highly diverse analogues in just two simple steps.

Immune Checkpoint PD-1/PD-L1: Is There Life Beyond Antibodies?

The PD-1/PD-L1 interaction has emerged as a significant target in cancer immunotherapy. Current medications include monoclonal antibodies, which have shown impressive clinical results in the treatment of several types of tumors. The cocrystal structure of human PD-1 and PD-L1 is expected to be a valuable starting point for the design of novel inhibitors, along with the recent crystal structures with monoclonal antibodies, small molecules, and macrocycles.

Synthesis and Pharmacochemistry of New Pleiotropic Pyrrolyl Derivatives.

Within the framework of our attempts to synthesize pleiotropic anti-inflammatory agents, we have synthesized some chalcones and their corresponding 3,4-pyrrolyl derivatives. Chalcones constitute a class of compounds with high biological impact. They are known for a number of biological activities, including anti-inflammatory and free radical scavenging activities. They inhibit several enzymes implicated in the inflammatory process, such as lipoxygenase, cyclooxygenase (COX) and lysozymes. The synthesized pyrroles have been studied for: (1) their in vitro inhibition of lipoxygenase; (2) their in vitro inhibition of COX; (3) their in vitro inhibition of lipid peroxidation; (4) their interaction with the stable, N-centered, free radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH); (5) their inhibition on interleukin-6 (IL-6); (6) their anti-proteolytic activity; and (7) their in vivo anti-inflammatory activity using carrageenan-induced rat paw edema. Their physicochemical properties were determined to explain the biological results. Lipophilicity was experimentally determined. 2i and 2v were found to be promising multifunctional molecules with high antiproteolytic and anti-inflammatory activities in combination with anti-interleukin-6 activity.

Meet the authors: Markella Konstantinidou and Michelle R. Arkin.

In an interview with Dr. Ying Li, a scientific editor of Cell Chemical Biology, the authors of the Review titled "Molecular glues for protein-protein interactions: Progressing toward a new dream" share their perspectives on their field and life as scientists.

Molecular glues for protein-protein interactions: Progressing toward a new dream.

The modulation of protein-protein interactions with small molecules is one of the most rapidly developing areas in drug discovery. In this review, we discuss advances over the past decade (2014-2023) focusing on molecular glues (MGs)-monovalent small molecules that induce proximity, either by stabilizing native interactions or by inducing neomorphic interactions. We include both serendipitous and rational discoveries and describe the different approaches that were used to identify them. We classify the compounds in three main categories: degradative MGs, non-degradative MGs or PPI stabilizers, and MGs that induce self-association. Diverse, illustrative examples with structural data are described in detail, emphasizing the elements of molecular recognition and cooperative binding at the interface that are fundamental for a MG mechanism of action.

Development of a NanoBRET assay for evaluation of 14-3-3σ molecular glues.

We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC50 values between 100 nM and 1 µM in cells, which align with the EC50 values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.

The tale of proteolysis targeting chimeras (PROTACs) for Leucine-Rich Repeat Kinase 2 (LRRK2).

Here we present the rational design and synthetic methodologies towards proteolysis-targeting chimeras (PROTACs) for the recently-emerged target leucine-rich repeat kinase 2 (LRRK2). Two highly potent, selective, brain-penetrating kinase inhibitors were selected, and their structure was appropriately modified to assemble a cereblon-targeting PROTAC. Biological data show strong kinase inhibition and the ability of the synthesized compounds to enter the cells. However, data regarding the degradation of the target protein are inconclusive. The reasons for the inefficient degradation of the target are further discussed.

Covalent inhibitors: a rational approach to drug discovery.

Covalent inhibitors are recognized as an important component in drug discovery and therapeutics. Since the first appearance of covalent inhibitors in the late 18th century, the field has advanced significantly and currently about 30% of the marketed drugs are covalent inhibitors. The numerous advantages of covalent inhibitors are counteracting the initial concerns regarding potential off-target toxicity. Thus, continuous research, especially for cancer targets is reported. The aim of this review is to provide a short historic overview and focus on recently developed covalent inhibitors (2011-2019), including structural aspects and examples on challenging targets.

Rapid Discovery of Aspartyl Protease Inhibitors Using an Anchoring Approach.

Pharmacophore searches that include anchors, fragments contributing above average to receptor binding, combined with one-step syntheses are a powerful approach for the fast discovery of novel bioactive molecules. Here, we are presenting a pipeline for the rapid and efficient discovery of aspartyl protease inhibitors. First, we hypothesized that hydrazine could be a multi-valent warhead to interact with the active site Asp carboxylic acids. We incorporated the hydrazine anchor in a multicomponent reaction and created a large virtual library of hydrazine derivatives synthetically accessible in one-step. Next, we performed anchor-based pharmacophore screening of the libraries and resynthesized top-ranked compounds. The inhibitory potency of the molecules was finally assessed by an enzyme activity assay and the binding mode confirmed by several soaked crystal structures supporting the validity of the hypothesis and approach. The herein reported pipeline of tools will be of general value for the rapid generation of receptor binders beyond Asp proteases.

1,3,4-Oxadiazoles by Ugi-Tetrazole and Huisgen Reaction.

Easy to perform, functional group tolerant, and short syntheses of the privileged scaffold oxadiazole are highly desired. Here, a metal-free protocol for MCR-based synthesis of 2,5-disubstituted 1,3,4-oxadiazoles via a Ugi-tetrazole/Huisgen sequence was developed. Optimization and scope and limitations of this short and general sequence are described. The reaction was also successfully performed on a gram scale.

PROTACs- a game-changing technology.

Introduction: Proteolysis - targeting chimeras (PROTACs) have emerged as a new modality with the potential to revolutionize drug discovery. PROTACs are heterobifunctional molecules comprising of a ligand targeting a protein of interest, a ligand targeting an E3 ligase and a connecting linker. The aim is instead of inhibiting the target to induce its proteasomal degradation. Areas covered: PROTACs, due to their bifunctional design, possess properties that differentiate them from classical inhibitors. A structural analysis, based on published crystal aspects, kinetic features and aspects of selectivity are discussed. Specific types such as homoPROTACs, PROTACs targeting Tau protein and the first PROTACs recently entering clinical trials are examined. Expert opinion: PROTACs have shown remarkable biological responses in challenging targets, including an unprecedented selectivity over protein family members and even efficacy starting from weak or unspecific binders. Moreover, PROTACs are standing out from classical pharmacology by inducing the degradation of the target protein and not merely its inhibition. However, there are also challenges in the field, such as the rational structure optimization, the evolution of computational tools, limited structural data and the greatly anticipated clinical data. Despite the remaining hurdles, PROTACs are expected to soon become a new therapeutic category of drugs.