All lysine acetyltransferases (KATs) modulate biological outcomes through the acetylation of lysine side-chain amino groups facilitated by acetyl coenzyme A (AcCoA). KAT6A belongs to the class of MYST domain histone acetyltransferases (HATs), which had been regarded as undruggable. The first on-target KAT6A inhibitors with in vivo activity were reported in 2018, catalyzing intense industry interest in this enzyme as an oncology target. In this study, we experimentally evaluated representative KAT6A inhibitor chemotypes through resynthesis and comparative biochemical assays, cellular assays, and structural biology. We outline the recent history of each KAT6A inhibitor chemotype discovery, including SAR for potency, selectivity, and cellular activity. We extensively benchmark key compounds from each chemotype, augmented by new acylsulfonohydrazide analogues and a novel fused [1,2,4]thiadiazine KAT6A inhibitor subclass, which we report here for the first time, along with co-crystal structures. Additionally, we report on the in vivo activity, pharmacokinetics, and toxicology profiles of these inhibitors.

Pain management costs the world billions of dollars each year, and there are limited nonopioid options to treat people suffering from chronic pain. Opioids are excellent analgesics but are liable to abuse and fatal overdoses. This Microperspective summarizes challenges and opportunities pertaining to creating nonopioid drugs that could be used to treat chronic pain, substance abuse, fatty liver, or obesity by targeting the cannabinoid receptor type 1 (CB1).

Sterol biosynthesis via the mevalonate-isoprenoid pathway produces ergosterol (24β-methyl cholesta-5,7-dienol) necessary for growth in a wide-range of eukaryotic pathogenic organisms in eukaryotes, including the fungi, trypanosomes and amoebae, while their animal hosts synthesize a structurally less complicated product—cholesterol (cholest-5-enol). Because phyla-specific differences in sterol metabolizing enzyme architecture governs the binding and reaction properties of substrates and inhibitors while the order of sterol metabolizing enzymes involved in steroidogenesis determine the positioning of crucial chokepoint enzymes in the biosynthetic pathway, the selectivity and effectiveness of rationally designed ergosterol biosynthesis inhibitors toward ergosterol-dependent infectious diseases varies greatly. Recent research has revealed an evolving toolbox of mechanistically distinct tight-binding inhibitors against two crucial methylation-demethylation biocatalysts—the C24 sterol methyl transferase (absent from humans) and the C14-sterol demethylase (present generally in humans and their eukaryotic pathogens). Importantly for rational drug design and development, the activities of these enzymes can be selectively blocked in ergosterol biosynthesis causing loss of ergosterol and cell killing without harm to the host organism. Here, we examine recent advances in our understanding of sterol biosynthesis and the reaction differences in catalysis for sterol methylation-demethylation enzymes across kingdoms. In addition, the novelties and nuances of structure-guided or mechanism-based approaches based on crystallographic mappings and substrate specificities of the relevant enzyme are contrasted to conventional phenotypic screening of small molecules as an approach to develop new and more effective pharmacological leads.

The World Health Organization (WHO) fungal priority pathogens list (WHO FPPL) published in 2022 highlighted the inequity and research challenges faced by researchers who study pathogenic fungi that afflict humans. Antifungal drugs are the only weapon available to treat infections; however, these drugs are old, are not effective against multidrug-resistant (MDR) fungal strains, and are associated with substantial toxicity in clinical use. This Microperspective summarizes challenges pertaining to antifungal drug discovery in addition to highlighting recent advances and antifungal agents in clinical trials.

Cholesterol biosynthesis, primarily associated with eukaryotes, occurs as an essential component of human metabolism with biosynthetic deregulation a factor in cancer viability. The segment that partitions between squalene and the C27-end cholesterol yields the main cholesterogenesis branch subdivided into the Bloch and Kandutsch-Russell pathways. Their importance in cell viability, in normal growth and development originates primarily from the amphipathic property and shape of the cholesterol molecule which makes it suitable as a membrane insert. Cholesterol can also convert to variant oxygenated product metabolites of distinct function producing a complex interplay between cholesterol synthesis and overall steroidogenesis. In this review, we disassociate the two sides of cholesterogenesisis affecting the type and amounts of systemic sterols—one which is beneficial to human welfare while the other dysfunctional leading to misery and disease that could result in premature death. Our focus here is first to examine the cholesterol biosynthetic genes, enzymes, and order of biosynthetic intermediates in human cholesterogenesis pathways, then compare the effect of proximal and distal inhibitors of cholesterol biosynthesis against normal and cancer cell growth and metabolism. Collectively, the inhibitor studies of druggable enzymes and specific biosynthetic steps, suggest a potential role of disrupted cholesterol biosynthesis, in coordination with imported cholesterol, as a factor in cancer development and as discussed some of these inhibitors have chemotherapeutic implications.

A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (1), a unique aryl acylsulfonohydrazide with an IC50 of 1.0 μM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as 55 and 80. These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 (3) as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly potent KAT6A inhibitor (IC50 = 6.3 nM; KD = 0.002 μM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs) 1 . Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10 ) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

A high-throughput screen for inhibitors of the histone acetyltransferase, KAT6A, led to identification of an aryl sulfonohydrazide derivative (CTX-0124143) that inhibited KAT6A with an IC50 of 1.0 μM. Elaboration of the structure-activity relationship and medicinal chemistry optimization led to the discovery of WM-8014 (97), a highly potent inhibitor of KAT6A (IC50 = 0.008 μM). WM-8014 competes with acetyl-CoA (Ac-CoA), and X-ray crystallographic analysis demonstrated binding to the Ac-CoA binding site. Through inhibition of KAT6A activity, WM-8014 induces cellular senescence and represents a unique pharmacological tool.

Primitive sterol evolution plays an important role in fossil record interpretation and offers potential therapeutic avenues for human disease resulting from nematode infections. Recognizing that C4-methyl stenol products [8(14)-lophenol] can be synthesized in bacteria while C4-methyl stanol products (dinosterol) can be synthesized in dinoflagellates and preserved as sterane biomarkers in ancient sedimentary rock is key to eukaryotic sterol evolution. In this regard, nematodes have been proposed to convert dietary cholesterol to 8(14)-lophenol by a secondary metabolism pathway that could involve sterol C4 methylation analogous to the C2 methylation of hopanoids (radicle-type mechanism) or C24 methylation of sterols (carbocation-type mechanism). Here, we characterized dichotomous cholesterol metabolic pathways in Caenorhabditis elegans that generate 3-oxo sterol intermediates in separate paths to lophanol (4-methyl stanol) and 8(14)-lophenol (4-methyl stenol). We uncovered alternate C3-sterol oxidation and Δ7 desaturation steps that regulate sterol flux from which branching metabolite networks arise, while lophanol/8(14)-lophenol formation is shown to be dependent on a sterol C4α-methyltransferse (4-SMT) that requires 3-oxo sterol substrates and catalyzes a newly discovered 3-keto-enol tautomerism mechanism linked to S-adenosyl-l-methionine-dependent methylation. Alignment-specific substrate-binding domains similarly conserved in 4-SMT and 24-SMT enzymes, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of methyl sterols. The combination of these results provides evolutionary leads to sterol diversity and points to cryptic C4-methyl steroidogenic pathways of targeted convergence that mediate lineage-specific adaptations.­­

Sterol 14α-demethylase (SDM) is essential for sterol biosynthesis and is the primary molecular target for clinical and agricultural antifungals. SDM has been demonstrated to be a valid drug target for antiprotozoal therapies, and much research has been focused on using SDM inhibitors to treat neglected tropical diseases such as human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis. Sterol C24-methyltransferase (24-SMT) introduces the C24-methyl group of ergosterol and is an enzyme found in pathogenic fungi and protozoa but is absent from animals. This difference in sterol metabolism has the potential to be exploited in the development of selective drugs that specifically target 24-SMT of invasive fungi or protozoa without adversely affecting the human or animal host. The synthesis and biological activity of SDM and 24-SMT inhibitors are reviewed herein.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function(1). Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)(2,3). KAT6A has essential roles in normal haematopoietic stem cells(4-6) and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia(7,8). Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers(8). KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus(9,10), a function that requires its KAT activity(10). Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days(11). These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells, and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism, but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription. We combined overexpression, RNAi, and genetic deletion of metabolic enzymes to study RORγ-dependent transcription. Our results are consistent with the RORγt ligand(s) being a cholesterol biosynthetic intermediate (CBI) downstream of lanosterol and upstream of zymosterol. Analysis of lipids bound to RORγ identified molecules with molecular weights consistent with CBIs. Furthermore, CBIs stabilized the RORγ ligand-binding domain and induced coactivator recruitment. Genetic deletion of metabolic enzymes upstream of the RORγt-ligand(s) affected the development of lymph nodes and Th17 cells. Our data suggest that CBIs play a role in lymphocyte development potentially through regulation of RORγt.

Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Here, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyl-transferase essential for sterol methylation and function of ergosterol, and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while having no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (k(cat)/k(inact) = 0.26 min(-1)/0.24 min(-1); partition ratio of 1.08), whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Delta(24)-sterol is a promising strategy for developing a new treatment for trypanosomiasis.

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24β-methyltransferase (TbSMT) and sterol 14α-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of TbSMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 μM from lipid-depleted media) with small amounts of ergosterol (1.2 μM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanism-based inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 μM each (ED50 values) resulting in parasite death. Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection.

We report a new strategy to develop low molecular weight (18-28 kDa) poly(N-acryloylmorpholine) (PNAM) polymers as bivalent inhibitors of cholera toxin (CT) using Reversible Addition-Fragmentation chain Transfer (RAFT) technology. The inhibitory activity of the galacto-conjugated polymers was examined (ELISA) and the series displayed moderate inhibitory activity (millimolar IC50).

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

We report the synthesis of a series of bivalent 1,2,3-triazole linked galactopyranosides as potential inhibitors of cholera toxin (CT). The inhibitory activity of the bivalent series was examined (ELISA) and the series showed low inhibitory activity (millimolar IC(50)s). Conversely, the monomeric galactotriazole analogues were strong inhibitors of cholera toxin (IC(50) = 71-75 μM).