Cyclic ß2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease.

Due to their constrained conformations, cyclic ß2,3-amino acids (cßAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cßAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cßAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating 3 kinds of cßAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (ß1), (1S,2S)-2-aminocyclohexane carboxylic acid (ß2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one ß2 and two ß1, exhibited potent inhibitory activities with IC50 values of 40 and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 and >168 h, respectively. Notably, BM3A and BM7A, wherein the cßAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cßAA to the activity and stability of peptides. Overall, our results highlight the potential of cßAA in generating potent and highly stable macrocyclic peptides with drug-like properties.

A Small-Molecule Inhibitor of Factor Inhibiting HIF Binding to a Tyrosine-flip Pocket for the Treatment of Obesity.

In animals limiting oxygen upregulates hypoxia-inducible factor (HIF) promoting a metabolic shift towards glycolysis. Factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that regulates HIF function by reducing its interaction with histone acetyl transferases. HIF levels are negatively regulated by the HIF prolyl hydroxylases (PHDs), which like FIH, are 2-oxoglutarate(2OG) oxygenases. Genetic loss of FIH promotes both glycolysis and aerobic metabolism. FIH has multiple non-HIF substrates making it challenging to connect its biochemistry with physiology. A structure-mechanism guided approach identified a highly potent in vivo active FIH inhibitor, ZG-2291, binding of which promotes a conformational flip of a catalytically important tyrosine, enabling selective inhibition of FIH over other JmjC subfamily 2OG oxygenases. Consistent with genetic studies, ZG-2291 promotes thermogenesis and ameliorates symptoms of obesity and metabolic dysfunction in ob/ob mice. The results reveal ZG-2291 as a useful probe for the physiological functions of FIH and identify FIH inhibition as a promising strategy for obesity treatment.

Thiophene-fused γ-lactams inhibit the SARS-CoV-2 main protease via reversible covalent acylation.

Enzyme inhibitors working by O-acylation of nucleophilic serine residues are of immense medicinal importance, as exemplified by the ß-lactam antibiotics. By contrast, inhibition of nucleophilic cysteine enzymes by S-acylation has not been widely exploited for medicinal applications. The SARS-CoV-2 main protease (Mpro) is a nucleophilic cysteine protease and a validated therapeutic target for COVID-19 treatment using small-molecule inhibitors. The clinically used Mpro inhibitors nirmatrelvir and simnotrelvir work via reversible covalent reaction of their electrophilic nitrile with the Mpro nucleophilic cysteine (Cys145). We report combined structure activity relationship and mass spectrometric studies revealing that appropriately functionalized γ-lactams can potently inhibit Mpro by reversible covalent reaction with Cys145 of Mpro. The results suggest that γ-lactams have potential as electrophilic warheads for development of covalently reacting small-molecule inhibitors of Mpro and, by implication, other nucleophilic cysteine enzymes.

The selective prolyl hydroxylase inhibitor IOX5 stabilizes HIF-1α and compromises development and progression of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.

Selective targeting of human TET1 by cyclic peptide inhibitors: Insights from biochemical profiling.

Ten-Eleven Translocation (TET) enzymes are Fe(II)/2OG-dependent oxygenases that play important roles in epigenetic regulation, but selective inhibition of the TETs is an unmet challenge. We describe the profiling of previously identified TET1-binding macrocyclic peptides. TiP1 is established as a potent TET1 inhibitor (IC50 = 0.26 µM) with excellent selectivity over other TETs and 2OG oxygenases. TiP1 alanine scanning reveals the critical roles of Trp10 and Glu11 residues for inhibition of TET isoenzymes. The results highlight the utility of the RaPID method to identify potent enzyme inhibitors with selectivity over closely related paralogues. The structure-activity relationship data generated herein may find utility in the development of chemical probes for the TETs.

Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate.

Ten-eleven translocation enzymes (TETs) are Fe(II)/2-oxoglutarate (2OG) oxygenases that catalyze the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in eukaryotic DNA. Despite their roles in epigenetic regulation, there is a lack of reported TET inhibitors. The extent to which 2OG oxygenase inhibitors, including clinically used inhibitors and oncometabolites, modulate DNA modifications via TETs has been unclear. Here, we report studies on human TET1-3 inhibition by a set of 2OG oxygenase-focused inhibitors, employing both enzyme-based and cellular assays. Most inhibitors manifested similar potencies for TET1-3 and caused increases in cellular 5hmC levels. (R)-2-Hydroxyglutarate, an oncometabolite elevated in isocitrate dehydrogenase mutant cancer cells, showed different degrees of inhibition, with TET1 being less potently inhibited than TET3 and TET2, potentially reflecting the proposed role of TET2 mutations in tumorigenesis. The results highlight the tractability of TETs as drug targets and provide starting points for selective inhibitor design.

Structure-guided optimisation of N-hydroxythiazole-derived inhibitors of factor inhibiting hypoxia-inducible factor-α.

The human 2-oxoglutarate (2OG)- and Fe(ii)-dependent oxygenases factor inhibiting hypoxia-inducible factor-α (FIH) and HIF-α prolyl residue hydroxylases 1-3 (PHD1-3) regulate the response to hypoxia in humans via catalysing hydroxylation of the α-subunits of the hypoxia-inducible factors (HIFs). Small-molecule PHD inhibitors are used for anaemia treatment; by contrast, few selective inhibitors of FIH have been reported, despite their potential to regulate the hypoxic response, either alone or in combination with PHD inhibition. We report molecular, biophysical, and cellular evidence that the N-hydroxythiazole scaffold, reported to inhibit PHD2, is a useful broad spectrum 2OG oxygenase inhibitor scaffold, the inhibition potential of which can be tuned to achieve selective FIH inhibition. Structure-guided optimisation resulted in the discovery of N-hydroxythiazole derivatives that manifest substantially improved selectivity for FIH inhibition over PHD2 and other 2OG oxygenases, including Jumonji-C domain-containing protein 5 (∼25-fold), aspartate/asparagine-ß-hydroxylase (>100-fold) and histone Nε-lysine demethylase 4A (>300-fold). The optimised N-hydroxythiazole-based FIH inhibitors modulate the expression of FIH-dependent HIF target genes and, consistent with reports that FIH regulates cellular metabolism, suppressed lipid accumulation in adipocytes. Crystallographic studies reveal that the N-hydroxythiazole derivatives compete with both 2OG and the substrate for binding to the FIH active site. Derivatisation of the N-hydroxythiazole scaffold has the potential to afford selective inhibitors for 2OG oxygenases other than FIH.

5-Substituted Pyridine-2,4-dicarboxylate Derivatives Have Potential for Selective Inhibition of Human Jumonji-C Domain-Containing Protein 5.

Jumonji-C domain-containing protein 5 (JMJD5) is a 2-oxoglutarate (2OG)-dependent oxygenase that plays important roles in development, circadian rhythm, and cancer through unclear mechanisms. JMJD5 has been reported to have activity as a histone protease, as an Nε-methyl lysine demethylase, and as an arginine residue hydroxylase. Small-molecule JMJD5-selective inhibitors will be useful for investigating its (patho)physiological roles. Following the observation that the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylic acid (2,4-PDCA) is a 2OG-competing JMJD5 inhibitor, we report that 5-aminoalkyl-substituted 2,4-PDCA derivatives are potent JMJD5 inhibitors manifesting selectivity for JMJD5 over other human 2OG oxygenases. Crystallographic analyses with five inhibitors imply induced fit binding and reveal that the 2,4-PDCA C5 substituent orients into the JMJD5 substrate-binding pocket. Cellular studies indicate that the lead compounds display similar phenotypes as reported for clinically observed JMJD5 variants, which have a reduced catalytic activity compared to wild-type JMJD5.

Kinetic and inhibition studies on human Jumonji-C (JmjC) domain-containing protein 5.

Jumonji-C (JmjC) domain-containing protein 5 (JMJD5) is a human 2-oxoglutarate (2OG) and Fe(ii)-dependent oxygenase which catalyses the post-translational C3 hydroxylation of arginyl-residues and which is linked to the circadian rhythm and to cancer biology through as yet unidentified mechanisms. We report robust solid phase extraction coupled to mass spectrometry (SPE-MS)-based JMJD5 assays which enable kinetic and high-throughput inhibition studies. The kinetic studies reveal that some synthetic 2OG derivatives, notably including a 2OG derivative with a cyclic carbon backbone (i.e. (1R)-3-(carboxycarbonyl)cyclopentane-1-carboxylic acid), are efficient alternative cosubstrates of JMJD5 and of factor inhibiting hypoxia-inducible transcription factor HIF-α (FIH), but not of the Jumonji-C (JmjC) histone Nε-methyl lysine demethylase KDM4E, apparently reflecting the closer structural similarity of JMJD5 and FIH. The JMJD5 inhibition assays were validated by investigating the effect of reported 2OG oxygenase inhibitors on JMJD5 catalysis; the results reveal that broad-spectrum 2OG oxygenase inhibitors are also efficient JMJD5 inhibitors (e.g. N-oxalylglycine, pyridine-2,4-dicarboxylic acid, ebselen) whereas most 2OG oxygenase inhibitors that are in clinical use (e.g. roxadustat) do not inhibit JMJD5. The SPE-MS assays will help enable the development of efficient and selective JMJD5 inhibitors for investigating the biochemical functions of JMJD5 in cellular studies.

In vitro selection of macrocyclic peptide inhibitors containing cyclic γ2,4-amino acids targeting the SARS-CoV-2 main protease.

γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1' catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.

Substrate selectivity and inhibition of histidine JmjC hydroxylases MINA53 and NO66.

Non-haem Fe(ii) and 2-oxoglutarate (2OG) dependent oxygenases catalyse oxidation of multiple proteins in organisms ranging from bacteria to humans. We describe studies on the substrate selectivity and inhibition of the human ribosomal oxygenases (ROX) MINA53 and NO66, members of the JmjC 2OG oxygenase subfamily, which catalyse C-3 hydroxylation of histidine residues in Rpl27a and Rpl8, respectively. Assays with natural and unnatural histidine analogues incorporated into Rpl peptides provide evidence that MINA53 and NO66 have narrow substrate selectivities compared to some other human JmjC hydroxylases, including factor inhibiting HIF and JMJD6. Notably, the results of inhibition assays with Rpl peptides containing histidine analogues with acyclic side chains, including Asn, Gln and homoGln, suggest the activities of MINA53/NO66, and by implication related 2OG dependent protein hydroxylases/demethylases, might be regulated in vivo by competition with non-oxidised proteins/peptides. The inhibition results also provide avenues for development of inhibitors selective for MINA53 and NO66.

Alkyne Derivatives of SARS-CoV-2 Main Protease Inhibitors Including Nirmatrelvir Inhibit by Reacting Covalently with the Nucleophilic Cysteine.

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.

Formaldehyde reacts with N-terminal proline residues to give bicyclic aminals.

Formaldehyde (HCHO) is a potent electrophile that is toxic above threshold levels, but which is also produced in the nuclei of eukaryotic cells by demethylases. We report studies with the four canonical human histones revealing that histone H2B reacts with HCHO, including as generated by a histone demethylase, to give a stable product. NMR studies show that HCHO reacts with the N-terminal proline and associated amide of H2B to give a 5,5-bicyclic aminal that is relatively stable to competition with HCHO scavengers. While the roles of histone modification by this reaction require further investigation, we demonstrated the potential of N-terminal aminal formation to modulate protein function by conducting biochemical and cellular studies on the effects of HCHO on catalysis by 4-oxalocrotonate tautomerase, which employs a nucleophilic N-terminal proline. The results suggest that reactions of N-terminal residues with HCHO and other aldehydes have potential to alter protein function.

The catalytic domains of all human KDM5 JmjC demethylases catalyse N-methyl arginine demethylation.

The demethylation of Nε -methyllysine residues on histones by Jumonji-C lysine demethylases (JmjC-KDMs) has been established. A subset of JmjC-KDMs has also been reported to have Nω -methylarginine residue demethylase (RDM) activity. Here, we describe biochemical screening studies, showing that the catalytic domains of all human KDM5s (KDM5A-KDM5D), KDM4E and, to a lesser extent, KDM4A/D, have both KDM and RDM activities with histone peptides. Ras GTPase-activating protein-binding protein 1 peptides were shown to be RDM substrates for KDM5C/D. No RDM activity was observed with KDM1A and the other JmjC-KDMs tested. The results highlight the potential of JmjC-KDMs to catalyse reactions other than Nε -methyllysine demethylation. Although our study is limited to peptide fragments, the results should help guide biological studies investigating JmjC functions.

Reading and erasing of the phosphonium analogue of trimethyllysine by epigenetic proteins.

N ε-Methylation of lysine residues in histones plays an essential role in the regulation of eukaryotic transcription. The 'highest' methylation mark, N ε-trimethyllysine, is specifically recognised by N ε-trimethyllysine binding 'reader' domains, and undergoes demethylation, as catalysed by 2-oxoglutarate dependent JmjC oxygenases. We report studies on the recognition of the closest positively charged N ε-trimethyllysine analogue, i.e. its trimethylphosphonium derivative (KPme3), by N ε-trimethyllysine histone binding proteins and Nε-trimethyllysine demethylases. Calorimetric and computational studies with histone binding proteins reveal that H3KP4me3 binds more tightly than the natural H3K4me3 substrate, though the relative differences in binding affinity vary. Studies with JmjC demethylases show that some, but not all, of them can accept the phosphonium analogue of their natural substrates and that the methylation state selectivity can be changed by substitution of nitrogen for phosphorus. The combined results reveal that very subtle changes, e.g. substitution of nitrogen for phosphorus, can substantially affect interactions between ligand and reader domains / demethylases, knowledge that we hope will inspire the development of highly selective small molecules modulating their activity.

Development and Characterization of Type I, Type II, and Type III LIM-Kinase Chemical Probes.

LIMKs are important regulators of actin and microtubule dynamics, and they play essential roles in many cellular processes. Deregulation of LIMKs has been linked to the development of diverse diseases, including cancers and cognitive disabilities, but well-characterized inhibitors known as chemical probes are still lacking. Here, we report the characterization of three highly selective LIMK1/2 inhibitors covering all canonical binding modes (type I/II/III) and the structure-based design of the type II/III inhibitors. Characterization of these chemical probes revealed a low nanomolar affinity for LIMK1/2, and all inhibitors 1 (LIMKi3; type I), 48 (TH470; type II), and 15 (TH257; type III) showed excellent selectivity in a comprehensive scanMAX kinase selectivity panel. Phosphoproteomics revealed remarkable differences between type I and type II inhibitors compared with the allosteric inhibitor 15. In phenotypic assays such as neurite outgrowth models of fragile X-chromosome, 15 showed promising activity, suggesting the potential application of allosteric LIMK inhibitors treating this orphan disease.

Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6.

The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.

Penicillin Derivatives Inhibit the SARS-CoV-2 Main Protease by Reaction with Its Nucleophilic Cysteine.

The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of ß-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mpro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that ß-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl-enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by ß-lactams and related acylating agents.

Structure-Activity Studies Reveal Scope for Optimisation of Ebselen-Type Inhibition of SARS-CoV-2 Main Protease.

The reactive organoselenium compound ebselen is being investigated for treatment of coronavirus disease 2019 (COVID-19) and other diseases. We report structure-activity studies on sulfur analogues of ebselen with the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro ), employing turnover and protein-observed mass spectrometry-based assays. The results reveal scope for optimisation of ebselen/ebselen derivative- mediated inhibition of Mpro , particularly with respect to improved selectivity.

2-Oxoglutarate derivatives can selectively enhance or inhibit the activity of human oxygenases.

2-Oxoglutarate (2OG) oxygenases are validated agrochemical and human drug targets. The potential for modulating their activity with 2OG derivatives has not been explored, possibly due to concerns regarding selectivity. We report proof-of-principle studies demonstrating selective enhancement or inhibition of 2OG oxygenase activity by 2-oxo acids. The human 2OG oxygenases studied, factor inhibiting hypoxia-inducible transcription factor HIF-α (FIH) and aspartate/asparagine-ß-hydroxylase (AspH), catalyze C3 hydroxylations of Asp/Asn-residues. Of 35 tested 2OG derivatives, 10 enhance and 17 inhibit FIH activity. Comparison with results for AspH reveals that 2OG derivatives selectively enhance or inhibit FIH or AspH. Comparison of FIH structures complexed with 2OG derivatives to those for AspH provides insight into the basis of the observed selectivity. 2-Oxo acid derivatives have potential as drugs, for use in biomimetic catalysis, and in functional studies. The results suggest that the in vivo activity of 2OG oxygenases may be regulated by natural 2-oxo acids other than 2OG.