Class I histone deacetylases (HDACs 1–3) serve as catalytic subunits within seven multiprotein co-repressor complexes, each of which has distinct functions in the cell. We report the synthesis of a HDAC inhibitor–nanogold probe, derived from the class I HDAC inhibitor CI-994, for cryo-electron microscopy (cryo-EM) visualization of the HDAC catalytic domain within class I HDAC co-repressor complexes. The nanogold probe retained HDAC inhibitory activity comparable to CI-994 against the HDAC1-LSD1-CoREST complex in vitro. In cryo-EM studies, 2D class averages revealed the bi-lobed architecture of the CoREST complex and partial localization of the gold nanoparticle probe to the CoREST complex. However, the probe was not observed in classes showing the side-view of the CoREST complex, limiting unambiguous identification and positioning of the HDAC catalytic domain within the CoREST complex.

Despite the good options for the management of Diffuse large B-cell lymphoma (DLBCL), a significant percentage of patients either do not respond to current treatments or relapse after a short time. Thus, a wider palette of targeted therapeutic strategies is needed. Histone deacetylases (HDACs) inhibitors have shown promising responses in B-cell malignancies, but their off-target effects limit their efficiency. Here, we investigated the use of novel targeted therapeutics against class I HDACs to specifically induce cell death in DLBCL cells. We show that a proteolysis targeting chimera (PROTAC) that combined HDAC inhibitor CI-994 and an IAP ligand had a strong effect in killing different DLBCL cell lines, being more effective in doing so than CI-994 on its own. Moreover, we show that this was concomitant with the induction of DNA damage and apoptosis. A proteomics screen showed that the mechanism of induction of cell death by this PROTAC likely depends on the simultaneous activation of pro-apoptotic proteins (such as PARP-1, PDCD6IP, DAPk1, TP53BP1, and CACYBP) and the inhibition of pro-survival pathways. We conclude that eliminating class I HDACs with specific PROTACs could be an effective and precise strategy for treating DLBCL that should be further tested for their potential clinical relevance. Trial Registration: The authors have confirmed clinical trial registration is not needed for this submission.

Class I histone deacetylases regulate gene transcription and are established therapeutic targets. HDAC1-3 form the catalytic subunit in several distinct multiprotein complexes; however, HDAC inhibitors are rarely studied in the context of these complexes. We evaluated multiple inhibitors, using seven HDAC complexes, and found that the inhibition profiles were highly complex-dependent, despite targeting the same enzyme. We also investigated the effect of the allosteric regulator inositol phosphate on these inhibitors. We observed very large, complex-selective reductions in the potency of benzamides bearing a "foot-pocket group", proposed to be selective for HDAC1/2. The potencies of these compounds are likely to be profoundly different in vivo compared with in vitro potencies in the absence of inositol phosphates. Our findings are supported by cell-based assays evaluating histone acetylation and HDAC degradation, highlighting the importance of evaluating HDACi in the context of HDAC complexes and inositol phosphates.

Lysine-specific histone demethylase 1A (LSD1) is involved in epigenetic regulation and is a viable drug target with a number of LSD1 inhibitors in clinical trials. We report synthetic and structure-activity studies of two LSD1 inhibitors, TCP and SP2577, in clinical trials towards PROTAC development. 16 Heterobifunctional molecules were synthesised based on TCP and SP2577. No LSD1 degraders were identified in HCT116 cells, however two TCP analogues functionalised from the phenyl ring with an aklyl and PEG linker in combination with a VHL ligand demonstrated potent LSD1 inhibition in vitro in the HDAC1-CoREST-LSD1 complex (43 nM and 63 nM respectively). Our findings provide important SAR data towards LSD1 PROTACs.

Histone deacetylase inhibitors (HDACi) are widely used in cancer therapy but often suffer from off-target effects due to their pan-inhibitory activity towards zinc-dependent enzymes. Vorinostat (SAHA), a hydroxamate-based HDACi, has been shown to lack isoform selectivity, potentially leading to unintended interactions with other metalloenzymes. Here, we report high-resolution crystal structures of SAHA bound to human carbonic anhydrase II (CA II) and a carbonic anhydrase IX (CA IX) active-site mimic. Structures determined at room temperature and 100 K revealed two distinct SAHA conformers in both CA II and the CA IX mimic, with the hydroxamate moiety displacing the zinc-bound water and adopting either a tetrahedral or pentahedral coordination to Zn2+. Differences in hydrophobic interactions were observed between CA II and the CA IX mimic due to the F131V amino-acid difference between the two enzymes. SwissDock modeling accurately predicted the SAHA binding orientations observed in crystallography. Thermal shift assays using nanoDSF showed minimal stabilization of either CA by SAHA, in contrast to the potent CA inhibitor acetazolamide. Binding-energy calculations suggest that SAHA may bind carbonic anhydrases with affinities comparable to its HDAC targets. These findings highlight potential off-target binding of SAHA to carbonic anhydrases, which may contribute to its clinical side effects. The results also suggest that hydroxamates may serve as a nonsulfonamide scaffold for novel CA inhibitors, although isoform selectivity remains a challenge.

Histone deacetylase (HDAC) enzymes 1-3 exist in several corepressor complexes and are viable drug targets. To date, proteolysis targeting chimeras (PROTACs) designed to target HDAC1-3 typically exhibit the selective degradation of HDAC3. Herein, we report cereblon-recruiting PROTACs that degrade HDAC1 with selectivity over HDAC3.

Fetal hemoglobin (HbF) induction is a well-known strategy for the treatment of hemoglobinopathies such as Sickle Cell Disease (SCD) and Thalassemia, worldwide life-threatening conditions. Increased levels of HbF are able to prevent HbS polymerization and mitigate disease manifestations, resulting in lower morbidity and mortality in SCD. Nowadays there are only four drugs approved by the FDA for SCD treatment: Hydroxyurea, voxelotor, L-glutamine and crizanlizumab, while the search for new effective and safe treatments are scarce. Controlling the expression of the gamma-globin gene by inhibiting or degrading epigenetic targets has been shown to be promising for HbF induction, and histone deacetylases (HDACs 1 and 2) stand out as potential targets. Targeted protein degradation (TPD) was explored in this work with the synthesis of new cereblon-based molecules able to anchor to cereblon and HDACs 1 and 2 simultaneously, leading to their dose dependent decrease via polyubiquitination and proteasome degradation. Six new cereblon derivatives containing a HDAC inhibitor subunit were designed, synthesized and evaluated in HCT-116 cells for HDAC 1, 2 and 3 degradation via quantitative western blot at concentrations ranging from 0.01 to 10 µM. The promising results showed that three of these compounds (ARP-26, ARP-37 and ARP-49) were able to degrade HDAC-1 more selectively, when compared to HDAC-2 and HDAC-3, achieving DC50 values of 2.5 µM. A derivative with no ability to bind to cereblon was also synthesized resulting in no degradation of HDACs 1-3, suggesting that these compounds act via cereblon binding. ARP-49 was then selected for further studies in HUDEP-2 cell culture at concentrations of 500 and 750nM, and incubation time of 72 and 96h. After the treatment, cells were stained with HbF-antibody and the percentage of HbF positive cells was assessed by flow cytometry. The quantification of HBG1/2 mRNA was obtained by qPCR. HUDEP-2 cells exhibit a basal level of HbF positive cells lower than 1%, then the results obtained from our compounds were normalized based on the control. At 72h, the concentrations of 500nM and 750nM were able to significantly increase the HBG1/2 mRNA levels by 26.9-fold and 35.5-fold, respectively (CTRL = 0.027 ± 0.006 arbitrary units (a.u.) vs 500nM = 0.73 ±0.25 a.u. and 750nM = 0.96 ± 0.11 a.u., p<0.0001, n=4). At 96h, both concentrations were also able to significantly induce the expression of the HBG1/2 genes by 34-fold and 44.5-fold, respectively (CTRL = 0.034 ± 0.012 a.u. vs 500nM = 1.12 ± 0.35 a.u. and 750nM = 1.52 ± 0.37 a.u., p<0.0001, n=4). HbF-positive cells at 72h presented a significantly increase of 7.5-fold and 9-fold at 500 and 750nM, respectively (CTRL = 0.58 ± 0.15 % vs 500nM = 4.43 ± 0.88 % and 750nM = 5.29 ± 1.52 %, p<0.0001, n=4), while at 96h the increase was 8.37-fold and 10.6-fold, respectively (CTRL = 0.65 ± 0.15 % vs 500nM = 5.44 ± 0.79 % and 750nM = 6.89 ± 1.05 %, p<0.0001, n=4). HUDEP-2 cells were also treated with HU at 100 µM, reaching its highest level of HBG1/2 expression at 72h (10-fold), when compared to control, and an increase in the HbF-positive cells population by 8.5-fold and 8.9-fold at 72h and 96h, respectively (CTRL 72h = 0.66 ± 0.23% vs HU 72h = 5.65 ± 0.59%, CTRL 96h = 0.64 ± 0.15% vs HU 96h = 5.73 ± 0.52%; p<0.0001, n=2). Compounds obtained in this study present a degradation selectivity towards HDAC-1 with no previous description in the literature. Furthermore, the ability to significantly induce HBG1/2 expression in HUDEP-2 cells was superior to the one seen for the standard SCD treatment, HU, with levels of HBG1/2 expression 2.6-fold higher (500nM, 72h), at a concentration 200-fold lower. These findings support that ARP-49 shows promising potential for further pre-clinical studies for increasing the production of HbF.

The class I histone deacetylase (HDAC) enzymes;HDAC1,2 and 3 form the catalytic engine of at least seven structurally distinct multiprotein complexes in cells. These molecular machines play a vital role in the regulation of chromatin accessibility and gene activity via the removal of acetyl moieties from lysine residues within histone tails. Their inhibition via small molecule inhibitors has beneficial effects in a number of disease types, including the clinical treatment of hematological cancers. We have previously reported a library of proteolysis targeting chimeras (PROTACs) incorporating a benzamide-based HDAC ligand (from CI-994), with an alkyl linker and ligand for the von Hippel-Lindau (VHL) E3 ubiquitin ligase that degrade HDAC1-3 at submicromolar concentrations. Here we report the addition of two novel PROTACs (JPS026 and JPS027), which utilize a ligand for the cellular inhibitor of apoptosis (IAP) family of E3 ligases. We found that both VHL (JPS004)- and IAP (JPS026)-based PROTACs degrade HDAC1-3 and induce histone acetylation to a similar degree. However, JPS026 is significantly more potent at inducing cell death in HCT116 cells than is JPS004. RNA sequencing analysis of PROTAC-treated HCT116 cells showed a distinct gene expression signature in which cell cycle and DNA replication machinery are repressed. Components of the mTORC1 and -2 complexes were also reduced, leading to an increase in FOXO3 and downstream target genes that regulate autophagy and apoptosis. In summary, a novel combination of HDAC and IAP ligands generates a PROTAC with a potent ability to stimulate apoptosis and differential gene expression in human cancer cells.

Histone deacetylases 1-3 (HDAC1, HDAC2, and HDAC3) and their associated corepressor complexes play important roles in regulating chromatin structure and gene transcription. HDAC enzymes are also validated drug targets for oncology and offer promise toward new drugs for neurodegenerative diseases and cardiovascular diseases. We synthesized four novel heterobifunctional molecules designed to recruit the mouse double minute 2 homologue (MDM2) E3 ligase to degrade HDAC1-3 utilizing the MDM2 inhibitor idasanutlin, known as proteolysis targeting chimeras (PROTACs). Idasanutlin inhibits the MDM2-P53 protein-protein interaction and is in clinical trials. Although two MDM2-recruiting heterobifunctional molecules reduced HDAC1 and HDAC2 abundance with complete selectivity over HDAC3 and reduced HDAC1/2 corepressor components LSD1 and SIN3A, we were surprised to observe that idasanutlin alone was also capable of this effect. This finding suggests an association between the MDM2 E3 ligase and HDAC1/2 corepressor complexes, which could be important for designing future dual/bifunctional HDAC- and MDM2-targeting therapeutics, such as PROTACs.

Over the past three decades, we have witnessed the progression of small molecule chemical probes designed to inhibit the catalytic active site of histone deacetylase (HDAC) enzymes into FDA approved drugs. However, it is only in the past five years we have witnessed the emergence of proteolysis targeting chimeras (PROTACs) capable of promoting the proteasome mediated degradation of HDACs. This is a field still in its infancy, however given the current progress of PROTACs in clinical trials and the fact that FDA approved HDAC drugs are already in the clinic, there is significant potential in developing PROTACs to target HDACs as therapeutics. Beyond therapeutics, PROTACs also serve important applications as chemical probes to interrogate fundamental biology related to HDACs via their unique degradation mode of action. In this review, we highlight some of the key findings to date in the discovery of PROTACs targeting HDACs by HDAC class and HDAC isoenzyme, current gaps in PROTACs to target HDACs and future outlooks.

Click chemistry was utilised to prepare a library of PROTACs based on entinostat a class I histone deacetylase (HDAC) inhibitor in clinical trials. A novel PROTAC JMC-137 was identified as a HDAC1/2 and HDAC3 degrader in HCT116 cells. However, potency was compromised compared to previously identified class I HDAC PROTACs highlighting the importance in the choice of HDAC ligand, functional group for linker attachment and positioning in PROTAC design.

Class I histone deacetylase (HDAC) enzymes 1, 2, and 3 organize chromatin as the catalytic subunits within seven distinct multiprotein corepressorcomplexes and are established drug targets. We report optimization studies ofbenzamide-based Von Hippel-Lindau (VHL) E3-ligase proteolysis targeting chimeras(PROTACs) and for the first time describe transcriptome perturbations resulting from these degraders. By modifying the linker and VHL ligand, we identified PROTACs7,9, and22with submicromolar DC50values for HDAC1 and/or HDAC3 in HCT116cells. A hook effect was observed for HDAC3 that could be negated by modifying the position of attachment of the VHL ligand to the linker. The more potent HDAC1/2degraders correlated with greater total differentially expressed genes and enhanced apoptosis in HCT116 cells. We demonstrate thatHDAC1/2 degradation by PROTACs correlates with enhanced global gene expression and apoptosis, important for the development of more efficacious HDAC therapeutics with reduced side effects

Trinucleotide repeat (TNR) expansions cause nearly 20 severe human neurological diseases which are currently untreatable. For some of these diseases, ongoing somatic expansions accelerate disease progression and may influence age of onset. This new knowledge emphasizes the importance of understanding the protein factors that drive expansions. Recent genetic evidence indicates that the mismatch repair factor MutSβ (Msh2-Msh3 complex) and the histone deacetylase HDAC3 function in the same pathway to drive triplet repeat expansions. Here we tested the hypothesis that HDAC3 deacetylates MutSβ and thereby activates it to drive expansions. The HDAC3-selective inhibitor RGFP966 was used to examine its biological and biochemical consequences in human tissue culture cells. HDAC3 inhibition efficiently suppresses repeat expansion without impeding canonical mismatch repair activity. Five key lysine residues in Msh3 are direct targets of HDAC3 deacetylation. In cells expressing Msh3 in which these lysine residues are mutated to arginine, the inhibitory effect of RGFP966 on expansions is largely bypassed, consistent with the direct deacetylation hypothesis. RGFP966 treatment does not alter MutSβ subunit abundance or complex formation but does partially control its subcellular localization. Deacetylation sites in Msh3 overlap a nuclear localization signal, and we show that localization of MutSβ is partially dependent on HDAC3 activity. Together, these results indicate that MutSβ is a key target of HDAC3 deacetylation and provide insights into an innovative regulatory mechanism for triplet repeat expansions. The results suggest expansion activity may be druggable and support HDAC3-selective inhibition as an attractive therapy in some triplet repeat expansion diseases.

Histone deacetylase (HDAC) enzymes play crucial roles in epigenetic gene expression and are an attractive therapeutic target. Five HDAC inhibitors have been approved for cancer treatment to date, however, clinical applications have been limited due to poor single-agent drug efficacy and side effects associated with a lack of HDAC isoform or complex selectivity. An emerging strategy aiming to address these limitations is the development of bifunctional HDAC therapeutics—single molecules comprising a HDAC inhibitor conjugated to another specificity targeting moiety. This review summarises the recent advancements in novel types of dual-targeting HDAC modulators, including proteolysis-targeting chimeras (PROTACs), with a focus on HDAC isoform and complex selectivity, and the future potential of such bifunctional molecules in achieving enhanced drug efficacy and therapeutic benefits in treating disease.

We have identified a proteolysis targeting chimera (PROTAC) of class I HDACs 1, 2 and 3. The most active degrader consists of a benzamide HDAC inhibitor, an alkyl linker, and the von Hippel-Lindau E3 ligand. Our PROTAC increased histone acetylation levels and compromised colon cancer HCT116 cell viability, establishing a degradation strategy as an alternative to class I HDAC inhibition.

Antibiotic resistance is a global health concern and a current threat to modern medicine and society. New strategies for antibiotic drug design and delivery offer a glimmer of hope in a currently limited pipeline of new antibiotics. One strategy involves conjugating iron-chelating microbial siderophores to an antibiotic or antimicrobial agent to enhance uptake and antibacterial potency. Cefiderocol (S-649266) is a promising cephalosporin-catechol conjugate currently in phase III clinical trials that utilizes iron-mediated active transport and demonstrates enhanced potency against multi-drug resistant (MDR) Gram-negative pathogens. Such molecules demonstrate that siderophore-antibiotic conjugates could be important future medicines to add to our antibiotic arsenal. This review is written in the context of the chemical design of siderophore-antibiotic conjugates focusing on the differing siderophore, linker, and antibiotic components that make up conjugates. We selected chemically distinct siderophore-antibiotic conjugates as exemplary conjugates, rather than multiple analogues, to highlight findings to date. The review should offer a general guide to the uninitiated in the molecular design of siderophore-antibiotic conjugates.

A series of analogues of Pseudonocardia sp. natural products were synthesized, which have been reported to possess potent antibacterial activity against Helicobacter pylori and induce growth defects in Escherichia coli and Staphylococcus aureus. Taking inspiration from a methodology used in our total synthesis of natural products, we applied this methodology to access analogues possessing bulky N-substituents, traditionally considered to be challenging scaffolds. Screening of the library provided valuable insights into the structure-activity relationship of the bacterial growth defects, and suggested that selectivity between bacterial species should be attainable. Furthermore, a structurally related series of analogues was observed to inhibit production of the virulence factor pyocyanin in the human pathogen Pseudomonas aeruginosa, which may be a result of their similarity to the Pseudomonas quinolone signal (PQS) quorum sensing autoinducer. This provided new insights regarding the effect of N-substitution in PQS analogues, which has been hitherto underexplored.

Siderophore–antibiotic conjugates consist of an antibiotic covalently linked by a tether to a siderophore. Such conjugates can demonstrate enhanced uptake and internalisation to the bacterial cell resulting in significantly reduced MIC values and extended spectrum of activity. Phenothiazines are a class of small molecules that have been identified as a potential treatment for multidrug resistant tuberculosis and latent TB. Herein we report the design and synthesis of the first phenothiazine–siderophore conjugate. A convergent synthetic route was developed whereby the functionalised phenothiazine component was prepared in four steps and the siderophore component also prepared in four steps. In M. smegmatis the functionalised phenothiazine demonstrated an equipotent MIC value in direct comparison to the parent phenothiazine from which it was derived. The final conjugate was synthesised by amide bond formation between the two components and global deprotection of the PMB protecting groups to unmask the catechol iron chelating groups of the siderophore. The synthesis is readily amenable to the preparation of analogues whereby the siderophore component of the conjugate can be modified. The route will be used to prepare a library of siderophore–phenothiazine conjugates for full biological evaluation of much needed new antibacterial agents.

Design, synthesis and application of PQS and HHQ probes for investigating quinolone quorum sensing pathways using photoaffinity labeling.

The synthesis of four quinolone natural products from the actinomycete Pseudonocardia sp. is reported. The key step involved a sp 2 –sp 3 Suzuki–Miyaura reaction between a common boronic ester lateral chain and various functionalised quinolone cores. The quinolones slowed growth of E. coli and S. aureus by inducing extended lag phases.