Natural and Synthetic Macrocyclic Inhibitors of the Histone Deacetylase Enzymes

Inhibition of histone deacetylase (HDAC) enzymes has emerged as a target for development of cancer chemotherapy. Four compounds have gained approval for clinical use by the Food and Drug Administration in the US, and several are currently in clinical trials. However, none of these compounds possesses particularly good isozyme selectivity, which would be a highly desirable feature in a tool compound. Whether selective inhibition of individual HDAC isozymes will provide improved drug candidates remains to be seen. Nevertheless, it has been speculated that using macrocyclic compounds to target HDAC enzymes might hold an advantage over the use of traditional hydroxamic‐acid‐containing inhibitors, which rely on chelation to the conserved active‐site zinc ion. Here we review the literature on macrocyclic HDAC inhibitors obtained from natural sources and on structure–activity relationship studies inspired by these molecules, as well as on efforts aimed at fully synthetic macrocyclic HDAC inhibitors.     

Cytochrome P450 Enzymes and Drug Metabolism in Humans

Human cytochrome P450 (CYP) enzymes, as membrane-bound hemoproteins, play important roles in the detoxification of drugs, cellular metabolism, and homeostasis. In humans, almost 80% of oxidative metabolism and approximately 50% of the overall elimination of common clinical drugs can be attributed to one or more of the various CYPs, from the CYP families 1–3. In addition to the basic metabolic effects for elimination, CYPs are also capable of affecting drug responses by influencing drug action, safety, bioavailability, and drug resistance through metabolism, in both metabolic organs and local sites of action. Structures of CYPs have recently provided new insights into both understanding the mechanisms of drug metabolism and exploiting CYPs as drug targets. Genetic polymorphisms and epigenetic changes in CYP genes and environmental factors may be responsible for interethnic and interindividual variations in the therapeutic efficacy of drugs. In this review, we summarize and highlight the structural knowledge about CYPs and the major CYPs in drug metabolism. Additionally, genetic and epigenetic factors, as well as several intrinsic and extrinsic factors that contribute to interindividual variation in drug response are also reviewed, to reveal the multifarious and important roles of CYP-mediated metabolism and elimination in drug therapy.     

Recent Advances in Neuraminidase Inhibitor Development as Anti-influenza Drugs

The recent emergence of the highly pathogenic H5N1 subtype of avian influenza virus (AIV) and of the new type of human influenza A (H1N1) have emphasized the need for the development of effective anti‐influenza drugs. Presently, neuraminidase (NA) inhibitors are widely used in the treatment and prophylaxis of human influenza virus infection, and tremendous efforts have been made to develop more potent NA inhibitors to combat resistance and new influenza viruses. In this review, we discuss the structural characteristics of NA catalytic domains and the recent developments of new NA inhibitors using structure‐based drug design strategies. These drugs include analogues of zanamivir, analogues of oseltamivir, analogues of peramivir, and analogues of aromatic carboxylic acid and present promising options for therapeutics or leads for further development of NA inhibitors that may be useful in the event of a future influenza pandemic.     

The Anticancer Drug Ellipticine Activated with Cytochrome P450 Mediates DNA Damage Determining Its Pharmacological Efficiencies: Studies with Rats,Hepatic Cytochrome P450 Reductase Null (HRN?) Mice and Pure Enzymes

Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b₅ alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b₅, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b₅, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.     

Synthesis and Structural Characterisation of Selective Non‐Carbohydrate‐Based Inhibitors of Bacterial Sialidases

The major human pathogen Streptococcus pneumoniae plays a key role in several disease states including septicaemia, meningitis and community‐acquired pneumonia. Although vaccines against S. pneumoniae are available as prophylactics, there remains a need to identify and characterise novel chemical entities that can treat the diseases caused by this pathogen. S. pneumoniae expresses three sialidases, enzymes that cleave sialic acid from carbohydrate‐based surface molecules. Two of these enzymes, NanA and NanB, have been implicated in the pathogenesis of S. pneumoniae and are considered to be validated drug targets. Here we report our studies on the synthesis and structural characterisation of novel NanB‐selective inhibitors that are inspired by the β‐amino‐sulfonic acid family of buffers.     

Effects of Ospemifene on Drug Metabolism Mediated by Cytochrome P450 Enzymes in Humans in Vitro and in Vivo

The objective of these investigations was to determine the possible effects of the novel selective estrogen receptor modulator, ospemifene, on cytochrome P450 (CYP)-mediated drug metabolism. Ospemifene underwent testing for possible effects on CYP enzyme activity in human liver microsomes and in isolated human hepatocytes. Based on the results obtained in vitro, three Phase 1 crossover pharmacokinetic studies were conducted in healthy postmenopausal women to assess the in vivo effects of ospemifene on CYP-mediated drug metabolism. Ospemifene and its main metabolites 4-hydroxyospemifene and 4′-hydroxyospemifene weakly inhibited a number of CYPs (CYP2B6, CYP2C9, CYP2C19, CYP2C8, and CYP2D6) in vitro. However, only CYP2C9 activity was inhibited by 4-hydroxyospemifene at clinically relevant concentrations. Induction of CYPs by ospemifene in cultured human hepatocytes was 2.4-fold or less. The in vivo studies showed that ospemifene did not have significant effects on the areas under the plasma concentration-time curves of the tested CYP substrates warfarin (CYP2C9), bupropion (CYP2B6) and omeprazole (CYP2C19), demonstrating that pretreatment with ospemifene did not alter their metabolism. Therefore, the risk that ospemifene will affect the pharmacokinetics of drugs that are substrates for CYP enzymes is low.     

Mammalian Cells Engineered To Produce New Steroids

Steroids can be difficult to modify through traditional organic synthesis methods, but many enzymes regio‐ and stereoselectively process a wide variety of steroid substrates. We tested whether steroid‐modifying enzymes could make novel steroids from non‐native substrates. Numerous genes encoding steroid‐modifying enzymes, including some bacterial enzymes, were expressed in mammalian cells by transient transfection and found to be active. We made three unusual steroids by stable expression, in HEK293 cells, of the 7α‐hydroxylase CYP7B1, which was selected because of its high native product yield. These cells made 7α,17α‐dihydroxypregnenolone and 7β,17α‐dihydroxypregnenolone from 17α‐hydroxypregnenolone and produced 11α,16α‐dihydroxyprogesterone from 16α‐hydroxyprogesterone. The last two products were the result of CYP7B1‐catalyzed hydroxylation at previously unobserved sites. A Rosetta docking model of CYP7B1 suggested that these substrates’ D‐ring hydroxy groups might prevent them from binding in the same way as the native substrates, bringing different carbon atoms close to the active ferryl oxygen atom. This new approach could potentially use other enzymes and substrates to produce many novel steroids for drug candidate testing.     

Direct Staudinger–Phosphonite Reaction Provides Methylphosphonamidates as Inhibitors of CE4 De‐N‐acetylases

De‐N‐acetylases of β‐(1→6)‐D ‐N‐acetylglucosamine polymers (PNAG) and β‐(1→4)‐D ‐N‐acetylglucosamine residues in peptidoglycan are attractive targets for antimicrobial agents. PNAG de‐N‐acetylases are necessary for biofilm formation in numerous pathogenic bacteria. Peptidoglycan de‐N‐acetylation facilitates bacterial evasion of innate immune defenses. To target these enzymes, transition‐state analogue inhibitors containing a methylphosphonamidate have been synthesized through a direct Staudinger–phosphonite reaction. The inhibitors were tested on purified PgaB, a PNAG de‐N‐acetylase from Escherichia coli, and PgdA, a peptidoglycan de‐N‐acetylase from Streptococcus pneumonia. Herein, we describe the most potent inhibitor of peptidoglycan de‐N‐acetylases reported to date (K_i=80 μM ). The minimal inhibition of PgaB observed provides insight into key structural and functional differences in these enzymes that will need to be considered during the development of future inhibitors.     

Perspectives on Inhibiting β‐Amyloid Aggregation through Structure‐Based Drug Design

Targeting β‐amyloid (Aβ) remains the most desired strategy in Alzheimer’s disease (AD) drug discovery research. Many peptides that specifically target Aβ aggregates are known, encompassing efforts from both industrial and academic research settings. However, in clinical terms, not much success has been gained with peptide research; in turn, small drug‐like molecules are already globally recognized as showing promise as an alternate approach. Aβ aggregation inhibitors are the most important part of the multifunctional drug design regimen for treating AD. Unfortunately, rational drug design approaches with small molecules are still in the initial stages. Herein we highlight, update, and elaborate on the structural anatomy of Aβ and known Aβ aggregation inhibitors in hopes of helping to optimize their use in structure‐based drug design approaches toward inhibitors with greater specificity. Furthermore, we present the first review of efforts to target a previously uncharacterized region of acetylcholinesterase: the N‐terminal 7–20 sub‐region, which was experimentally elucidated to participate in Aβ aggregation and deposition.     

A Comprehensive Structural Overview of p38α Mitogen‐Activated Protein Kinase in Complex with ATP‐Site and Non‐ATP‐Site Binders

Herein we review all the currently available ATP‐site and non‐ATP‐site ligands bound to p38α mitogen‐activated protein kinase (MAPK) available in the RCSB Protein Data Bank (PDB). The co‐crystallized inhibitors have been classified into different families according to their experimental binding mode and chemical structure, and the ligand–protein interactions are discussed using the most representative compounds. This systematic structural analysis could provide some take‐home lessons for drug discovery programs aimed at the rational identification and optimization of new p38α MAPK inhibitors.     

Mechanisms of Enzyme-Catalyzed Reduction of Two Carcinogenic Nitro-Aromatics, 3-Nitrobenzanthrone and Aristolochic Acid I: Experimental and Theoretical Approaches

This review summarizes the results found in studies investigating the enzymatic activation of two genotoxic nitro-aromatics, an environmental pollutant and carcinogen 3-nitrobenzanthrone (3-NBA) and a natural plant nephrotoxin and carcinogen aristolochic acid I (AAI), to reactive species forming covalent DNA adducts. Experimental and theoretical approaches determined the reasons why human NAD(P)H:quinone oxidoreductase (NQO1) and cytochromes P450 (CYP) 1A1 and 1A2 have the potential to reductively activate both nitro-aromatics. The results also contributed to the elucidation of the molecular mechanisms of these reactions. The contribution of conjugation enzymes such as N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) to the activation of 3-NBA and AAI was also examined. The results indicated differences in the abilities of 3-NBA and AAI metabolites to be further activated by these conjugation enzymes. The formation of DNA adducts generated by both carcinogens during their reductive activation by the NOQ1 and CYP1A1/2 enzymes was investigated with pure enzymes, enzymes present in subcellular cytosolic and microsomal fractions, selective inhibitors, and animal models (including knock-out and humanized animals). For the theoretical approaches, flexible in silico docking methods as well as ab initio calculations were employed. The results summarized in this review demonstrate that a combination of experimental and theoretical approaches is a useful tool to study the enzyme-mediated reaction mechanisms of 3-NBA and AAI reduction.     

Functional Investigations of Thromboxane Synthase (CYP5A1) in Lipid Bilayers of Nanodiscs

CYP5A1 is a membrane‐associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH₂) into thromboxane A₂ (TXA₂), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER‐bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug‐metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small‐molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.     

Expanding the Binding Envelope of CYP51 Inhibitors Targeting Trypanosoma cruzi with 4‐Aminopyridyl‐Based Sulfonamide Derivatives

Chagas disease is a chronic infection caused by the protozoan parasite Trypanosoma cruzi, manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Therapeutic options to prevent or treat Chagas disease are limited. CYP51, the enzyme key to the biosynthesis of eukaryotic membrane sterols, is a validated drug target in both fungi and T. cruzi. Sulfonamide derivatives of 4‐aminopyridyl‐based inhibitors of T. cruzi CYP51 (TcCYP51), including the sub‐nanomolar compound 3 , have molecular structures distinct from other validated CYP51 inhibitors. They augment the biologically relevant chemical space of molecules targeting TcCYP51. In a 2.08 Å X‐ray structure, TcCYP51 is in a conformation that has been influenced by compound 3 and is distinct from the previously characterized ground‐state conformation of CYP51 drug–target complexes. That the binding site was modulated in response to an incoming inhibitor for the first time characterizes TcCYP51 as a flexible target rather than a rigid template.     

Selective Inhibitors of Glutathione Transferase P1 with Trioxane Structure as Anticancer Agents

The response to chemotherapy in cancer patients is frequently compromised by drug resistance. Although chemoresistance is a multifactorial phenomenon, many studies have demonstrated that altered drug metabolism through the expression of phase II conjugating enzymes, including glutathione transferases (GSTs), in tumor cells can be directly correlated with resistance against a wide range of marketed anticancer drugs. In particular, overexpression of glutathione transferase P1 (GSTP1) appears to be a factor for poor prognosis during cancer therapy. Former and ongoing clinical trials have confirmed GSTP1 inhibition as a principle for antitumor therapy. A new series of 1,2,4‐trioxane GSTP1 inhibitors were designed via a type II photooxygenation route of allylic alcohols followed by acid‐catalyzed peroxyacetalization with aldehydes. A set of novel inhibitors exhibit low micromolar to high nanomolar inhibition of GSTP1, revealing preliminary SAR for further lead optimization. Importantly, high selectivity over another two human GST classes (GSTA1 and GSTM2) has been achieved. The trioxane GSTP1 inhibitors may therefore serve as a basis for the development of novel drug candidates in overcoming chemoresistance.     

GSK-3β, FYN,and DYRK1A: Master Regulators in Neurodegenerative Pathways

Protein kinases (PKs) have been recognized as central nervous system (CNS)-disease-relevant targets due to their master regulatory role in different signal transduction cascades in the neuroscience space. Among them, GSK-3β, FYN, and DYRK1A play a crucial role in the neurodegeneration context, and the deregulation of all three PKs has been linked to different CNS disorders with unmet medical needs, including Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal lobar degeneration (FTLD), and several neuromuscular disorders. The multifactorial nature of these diseases, along with the failure of many advanced CNS clinical trials, and the lengthy approval process of a novel CNS drug have strongly limited the CNS drug discovery. However, in the near-decade from 2010 to 2020, several computer-assisted drug design strategies have been combined with synthetic efforts to develop potent and selective GSK-3β, FYN, and DYRK1A inhibitors as disease-modifying agents. In this review, we described both structural and functional aspects of GSK-3β, FYN, and DYRK1A and their involvement and crosstalk in different CNS pathological signaling pathways. Moreover, we outlined attractive medicinal chemistry approaches including multi-target drug design strategies applied to overcome some limitations of known PKs inhibitors and discover improved modulators with suitable blood–brain barrier (BBB) permeability and drug-like properties.     

Metabolism of (+)-terpinen-4-ol by cytochrome P450 enzymes in human liver microsomes

We examined the in vitro metabolism of (+)-terpinen-4-ol by human liver microsomes and recombinant enzymes. The biotransformation of (+)-terpinen-4-ol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Terpinen-4-ol was found to be oxidized to (+)-(1R,2S,4S)-1,2-epoxy-p-menthan-4-ol, (+)-(1S,2R,4S)-1,2-epoxy-p-menthan-4-ol, and (4S)-p-menth-1-en-4,8-diol by human liver microsomal P450 enzymes. The identities of (+)-terpinen-4-ol metabolites were determined through the relative abundance of mass fragments and retention times on GC-MS. Of 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6, and CYP3A4 were found to catalyze the oxidation of (+)-terpinen-4-ol. Based on several lines of evidence, CYP2A6 and CYP3A4 were determined to be major enzymes involved in the oxidation of (+)-terpinen-4-ol by human liver microsomes. First, of the 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6 and CYP3A4 catalyzed oxidation of (+)-terpinen-4-ol. Second, oxidation of (+)-terpinen-4-ol was inhibited by (+)-menthofuran and ketoconazole, inhibitors known to be specific for these enzymes. Finally, there was a good correlation between CYP2A6 and CYP3A4 activities and (+)-terpinen-4-ol oxidation activities in the 10 human liver microsomes.     

Investigation of Binding‐Site Homology between Mushroom and Bacterial Tyrosinases by Using Aurones as Effectors

Tyrosinase is a copper‐containing enzyme found in plants and bacteria, as well as in humans, where it is involved in the biosynthesis of melanin‐type pigments. Tyrosinase inhibitors have attracted remarkable research interest as whitening agents in cosmetology, antibrowning agents in food chemistry, and as therapeutics. In this context, commercially available tyrosinase from mushroom (TyM) is frequently used for the identification of inhibitors. This and bacterial tyrosinase (TyB) have been the subjects of intense biochemical and structural studies, including X‐ray diffraction analysis, and this has led to the identification of structural homology and divergence among enzymes from different sources. To better understand the behavior of potential inhibitors of TyM and TyB, we selected the aurone family—previously identified as potential inhibitors of melanin biosynthesis in human melanocytes. In this study, a series of 24 aurones with different hydroxylation patterns at the A‐ and B‐rings were evaluated on TyM and TyB. The results show that, depending on the hydroxylation pattern of A‐ and B‐rings, aurones can behave as inhibitors, substrates, and activators of both enzymes. Computational analysis was performed to identify residues surrounding the aurones in the active sites of both enzymes and to rationalize the interactions. Our results highlight similarities and divergence in the behavior of TyM and TyB toward the same set of molecules.     

Induced‐Fit Docking Approach Provides Insight into the Binding Mode and Mechanism of Action of HIV‐1 Integrase Inhibitors

A three‐dimensional model of a complex between HIV‐1 integrase (IN), viral DNA, and metal ions that we recently built was used as a target for a docking method (induced‐fit docking, IFD) that accurately predicts ligand binding modes and concomitant structural changes in the receptor. Six different well‐known integrase strand transfer inhibitors (INSTIs): L‐708,906, L‐731,988, S‐1360, L‐870,810, raltegravir, and elvitegravir were thus used as ligands for our docking simulations. The obtained IFD results are consistent with the mechanism of action proposed for this class of IN inhibitors, that is, metal chelating/binding agents. This study affords new insight into the possible mechanism of inhibition and binding conformations for INSTIs. The impact on our hypothesis of specific mutations associated with IN inhibitor resistance was also evaluated. All these findings might have implications for integrase‐directed HIV‐1 drug discovery efforts.     

Chronic Inflammatory Status Observed in Patients with Type 2 Diabetes Induces Modulation of Cytochrome P450 Expression and Activity

Diabetes mellitus is a metabolic disease that causes a hyperglycemic status which leads, over time, to serious damage to the heart, blood vessels, eyes, kidneys and nerves. The most frequent form of diabetes is type 2 diabetes mellitus (T2DM) which is often part of a metabolic syndrome (hyperglycaemia, hypertension, hypercholesterolemia, abdominal obesity) that usually requires the use of several medications from different drug classes to bring each of these conditions under control. T2DM is associated with an increase in inflammatory markers such as interleukin-6 (IL-6) and the tumor necrosis factor alpha (TNF-α). Higher levels of IL-6 and TNF-α are associated with a downregulation of several drug metabolizing enzymes, especially the cytochrome P450 (P450) isoforms CYP3As and CYP2C19. A decrease in these P450 isoenzymes may lead to unexpected rise in plasma levels of substrates of these enzymes. It could also give rise to a mismatch between the genotypes determined for these enzymes, the predicted phenotypes based on these genotypes and the phenotypes observed clinically. This phenomenon is described as phenoconversion. Phenoconversion typically results from either a disease (such as T2DM) or concomitant administration of medications inducing or inhibiting (including competitive or non-competitive inhibition) a P450 isoenzyme used by other substrates for their elimination. Phenoconversion could have a significant impact on drug effects and genotypic-focused clinical outcomes. As the aging population is exposed to polypharmacy along with inflammatory comorbidities, consideration of phenoconversion related to drug metabolizing enzymes is of importance when applying pharmacogenomic results and establishing personalized and more precise drug regimens.     

Identification of HDAC6‐Selective Inhibitors of Low Cancer Cell Cytotoxicity

The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform‐selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first‐generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell‐cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole‐based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1–3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.