Farnesyltransferase inhibition in HGPS.

The ultra-rare, pediatric premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS) is caused by mutation of LMNA, encoding the nuclear architectural protein lamin A. Patients develop atherosclerosis and typically die of heart failure in their teens. FDA-approved Zokinvy prevents farnesylation of lamin A, reduces vascular stiffness, and extends survival in HGPS patients. To view this Bench to Bedside, open or download the PDF.

Structural basis of γ-secretase inhibition and modulation by small molecule drugs.

Development of γ-secretase inhibitors (GSIs) and modulators (GSMs) represents an attractive therapeutic opportunity for Alzheimer's disease (AD) and cancers. However, how these GSIs and GSMs target γ-secretase has remained largely unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human γ-secretase bound individually to two GSI clinical candidates, Semagacestat and Avagacestat, a transition state analog GSI L685,458, and a classic GSM E2012, at overall resolutions of 2.6-3.1 Å. Remarkably, each of the GSIs occupies the same general location on presenilin 1 (PS1) that accommodates the ß strand from amyloid precursor protein or Notch, interfering with substrate recruitment. L685,458 directly coordinates the two catalytic aspartate residues of PS1. E2012 binds to an allosteric site of γ-secretase on the extracellular side, potentially explaining its modulating activity. Structural analysis reveals a set of shared themes and variations for inhibitor and modulator recognition that will guide development of the next-generation substrate-selective inhibitors.

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

Polyamine metabolism is a central determinant of helper T cell lineage fidelity.

Polyamine synthesis represents one of the most profound metabolic changes during T cell activation, but the biological implications of this are scarcely known. Here, we show that polyamine metabolism is a fundamental process governing the ability of CD4+ helper T cells (TH) to polarize into different functional fates. Deficiency in ornithine decarboxylase, a crucial enzyme for polyamine synthesis, results in a severe failure of CD4+ T cells to adopt correct subset specification, underscored by ectopic expression of multiple cytokines and lineage-defining transcription factors across TH cell subsets. Polyamines control TH differentiation by providing substrates for deoxyhypusine synthase, which synthesizes the amino acid hypusine, and mice in which T cells are deficient for hypusine develop severe intestinal inflammatory disease. Polyamine-hypusine deficiency caused widespread epigenetic remodeling driven by alterations in histone acetylation and a re-wired tricarboxylic acid (TCA) cycle. Thus, polyamine metabolism is critical for maintaining the epigenome to focus TH cell subset fidelity.

Epigenetic Therapy for Epithelioid Sarcoma.

Tazemetostat is the first epigenetic therapy to gain FDA approval in a solid tumor. This lysine methyltransferase inhibitor targets EZH2, the enzymatic subunit of the PRC2 transcriptional silencing complex. Tumors with mutations in subunits of the SWI/SNF chromatin remodeling complex, inclusive of most epithelioid sarcomas, are sensitive to EZH2 inhibition.

Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design.

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.

Target-Based Discovery of an Inhibitor of the Regulatory Phosphatase PPP1R15B.

Protein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington's disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.

KRAS Dimerization Impacts MEK Inhibitor Sensitivity and Oncogenic Activity of Mutant KRAS.

The mechanism by which the wild-type KRAS allele imparts a growth inhibitory effect to oncogenic KRAS in various cancers, including lung adenocarcinoma (LUAD), is poorly understood. Here, using a genetically inducible model of KRAS loss of heterozygosity (LOH), we show that KRAS dimerization mediates wild-type KRAS-dependent fitness of human and murine KRAS mutant LUAD tumor cells and underlies resistance to MEK inhibition. These effects are abrogated when wild-type KRAS is replaced by KRASD154Q, a mutant that disrupts dimerization at the α4-α5 KRAS dimer interface without changing other fundamental biochemical properties of KRAS, both in vitro and in vivo. Moreover, dimerization has a critical role in the oncogenic activity of mutant KRAS. Our studies provide mechanistic and biological insights into the role of KRAS dimerization and highlight a role for disruption of dimerization as a therapeutic strategy for KRAS mutant cancers.

Mitochondrial Priming by CD28.

T cell receptor (TCR) signaling without CD28 can elicit primary effector T cells, but memory T cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon T cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for T cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory T cells. Our data show that initial CD28 signals during T cell activation prime mitochondria with latent metabolic capacity that is essential for future T cell responses.

Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis.

All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes.

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

A Synergistic Interaction between Chk1- and MK2 Inhibitors in KRAS-Mutant Cancer.

KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers.

Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

Enzyme inhibitor discovery by activity-based protein profiling.

Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.

Cellular pathways influenced by protein arginine methylation: Implications for cancer.

Arginine methylation is an influential post-translational modification occurring on histones, RNA binding proteins, and many other cellular proteins, affecting their function by altering their protein-protein and protein-nucleic acid interactions. Recently, a wealth of information has been gathered, implicating protein arginine methyltransferases (PRMTs), enzymes that deposit arginine methylation, in transcription, pre-mRNA splicing, DNA damage signaling, and immune signaling with major implications for cancer therapy, especially immunotherapy. This review summarizes this recent progress and the current state of PRMT inhibitors, some in clinical trials, as promising drug targets for cancer.

Structure, mechanism, and inhibition of Hedgehog acyltransferase.

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

A proteomic and phosphoproteomic landscape of KRAS mutant cancers identifies combination therapies.

KRAS mutant cancer, characterized by the activation of a plethora of phosphorylation signaling pathways, remains a major challenge for cancer therapy. Despite recent advancements, a comprehensive profile of the proteome and phosphoproteome is lacking. This study provides a proteomic and phosphoproteomic landscape of 43 KRAS mutant cancer cell lines across different tissue origins. By integrating transcriptomics, proteomics, and phosphoproteomics, we identify three subsets with distinct biological, clinical, and therapeutic characteristics. The integrative analysis of phosphoproteome and drug sensitivity information facilitates the identification of a set of drug combinations with therapeutic potentials. Among them, we demonstrate that the combination of DOT1L and SHP2 inhibitors is an effective treatment specific for subset 2 of KRAS mutant cancers, corresponding to a set of TCGA clinical tumors with the poorest prognosis. Together, this study provides a resource to better understand KRAS mutant cancer heterogeneity and identify new therapeutic possibilities.

Protein prenylation: unique fats make their mark on biology.

The modification of eukaryotic proteins by isoprenoid lipids, which is known as prenylation, controls the localization and activity of a range of proteins that have crucial functions in biological regulation. The roles of prenylated proteins in cells are well conserved across species, underscoring the biological and evolutionary importance of this lipid modification pathway. Genetic suppression and pharmacological inhibition of the protein prenylation machinery have provided insights into several cellular processes and into the aetiology of diseases in which prenylation is involved. The functional dependence of prenylation substrates, such as RAS proteins, on this modification and the therapeutic potential of targeting the prenylation process in pathological conditions accentuate the need to fully understand this form of post-translational modification.