Reviews & Analysis

We present a discovery pipeline integrating chemical fragment screening and time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS). This approach identifies allosteric chemical leads targeting distinct allosteric states of the mitochondrial oxidoreductase apoptosis-inducing factor (AIF). By monitoring kinetic rates of allosteric transition with TR-HT-SAXS, we link fragment structure–activity relationships (SARs) to biomolecular conformation.

The rate of ATP production and the total mass of enzymes were quantified for both glycolysis and mitochondrial respiration to determine the proteome efficiency of these pathways. Per unit of enzyme mass, mitochondrial respiration generates energy faster than glycolysis and is thus more proteome efficient. Despite being less proteome efficient, constitutive glycolysis comes with the benefit of rendering cells robust to hypoxia.

O-linked N-acetylglucosamine (O-GlcNAc) is an endogenous form of glycosylation that alters the structure of α-synuclein amyloid fibrils and attenuates their pathogenetic properties. The modified fibrils have a significantly reduced ability to seed the aggregation of endogenous α-synuclein in cultured neurons and in mice brains in vivo, which results in reduced pathology.

Nonribosomal peptide synthetases produce diverse natural products, including many valuable therapeutics. Although the condensation domains that catalyze peptide bond formation in these multifunctional enzymes have been difficult to engineer, a yeast display system that was developed to screen millions of variants now enables efficient reprogramming of synthetase substrate specificity.

SRI-41315 is a small molecule that enhances premature read-through of the stop codon by triggering the degradation of the translation termination factor eRF1. Cryo-EM structures and biochemical analyses reveal how SRI-41315 acts as a molecular glue between eRF1 and the decoding center of ribosomes, leading to increased recognition of the cryptic stop codon and eRF1 degradation.

Cyclic peptides can bind challenging disease targets, but their oral application is hindered by digestion and absorption issues. We developed a versatile method for the synthesis and functional screening of vast numbers of synthetic cyclic peptides and identified peptides with high inhibitory activity, stability and oral bioavailability in rats.

The requirement for a protospacer adjacent motif (PAM) is a well-known limitation of the CRISPR–Cas9 system, as it restricts the range of sequences that can be targeted. To address this limitation, we demonstrate a phage-assisted evolution approach for engineering a compact SlugCas9, simplifying its PAM requirement and broadening its DNA targeting scope.

Cells contain compartments composed of phase-separated protein condensates. We find that these condensates have a unique chemical microenvironment that enriches amphipathic metabolites such as phospholipids. Therefore, condensates are mixtures of proteins, nucleic acids and specific metabolites. The presence of phospholipids and other amphipathic metabolites might enable condensates to facilitate specific metabolic reactions.

LIS1 is an essential cofactor for the assembly of the cytoplasmic dynein transport machinery. How LIS1 binding affects dynein motility was unclear. Single-molecule experiments reveal that Pac1 (the yeast homolog of LIS1) binding reduces dynein speed by slowing its detachment from microtubules and does not disrupt the mechanism by which it generates force.

We developed a versatile lipid probe — MAO–SiR — to visualize the structure and dynamics of the inner mitochondrial membrane (IMM). MAO–SiR assembles in situ from two cell-permeant small molecules to image the IMM selectively, continuously and at super resolution for extended periods of time without extensive photobleaching or toxicity.

By investigating the structure–activity relationship of molecular glue degraders that target cyclin K, we discovered that a wide range of compounds, including known kinase inhibitors, possess this gain-of-function activity. These findings provide insights that might enable more rational design and optimization of molecular glue compounds.

Synthetic cells are modular gene-expressing compartments with promising applications in biology and medicine. However, a more diverse toolkit is needed to enhance their capabilities, particularly in terms of controlling their gene expression and employing novel synthetic cell–to–living cell signaling pathways. In this work, photocaged promoters and cell-free synthesis of the acyl homoserine lactone synthase BjaI were used to achieve light-activated communication between synthetic cells and living cells.

We designed FAK-SPARK, a fluorescent reporter of focal adhesion kinase (FAK) activity based on phase separation. FAK-SPARK revealed polarized FAK activity within single focal adhesions in the leading edges of migrating cells. By combining FAK-SPARK with DNA tension probes, we showed that FAK activity is proportional to the tension strength.

We identified a comprehensive targetome for glycolytic metabolites in cancer cells using a novel target discovery approach — target responsive accessibility profiling (TRAP). The targetome revealed diverse regulatory modalities for glycolytic metabolites that include engaging with metabolic enzymes, influencing transcriptional outputs and modulating post-translational modification levels, thereby elucidating how glycolytic metabolites function as signaling molecules.

We used chemical proteomics to identify candidate protein targets of indole metabolites in mammalian cells. We discovered that microbiota-derived and synthetic aromatic monoamines can activate recruitment of β-arrestin to the orphan receptor GPRC5A. Specific microbiota species that express amino acid decarboxylases were found to produce aromatic monoamine agonists for GPRC5A.

Protein stability is important for biological function, but little is known about in-cell stability. In the New Delhi metallo-β-lactamase NDM-1, enhancement of zinc binding or amino acid substitutions at the C terminus increase in-cell kinetic stability and prevent proteolysis. These findings link NDM-1-mediated resistance with its in-cell stability and physiology.

We identified small molecules that rewire the transcriptional state of cancer cells by covalently targeting the RNA-binding protein NONO. These small molecules stabilize the interactions of NONO with its target mRNAs, thereby overriding the compensatory action of paralog proteins and revealing a pharmacological strategy for disrupting previously undruggable oncogenic pathways.

Creatine kinases (CKs) have emerged as a metabolic liability in many rapidly proliferating cancers. We have developed a class of covalent inhibitors that impair creatine phosphagen energetics by targeting a redox-regulated cysteine residue in the active site of CKs.

Ferroptosis can be induced by lipid peroxidation in various subcellular membranes, including the endoplasmic reticulum (ER), mitochondria and lysosomes. By studying the subcellular distribution of ferroptosis-modulating fatty acids, we observed that the ER is a key initial site of peroxidation, followed by the plasma membrane, whereas other organelles are not as critical for ferroptosis.

Etoposide, a chemotherapeutic poison of type IIA eukaryotic topoisomerases (topo IIs), promotes topo II to compact DNA by trapping DNA loops, creates DNA double-strand breaks, causes topo II to resist relocation, and pauses the ability of topoisomerases to relax DNA supercoiling. Through these mechanisms, etoposide converts topo II into a roadblock to DNA processing.