Inhibition of Cysteine Proteases via Thiol-Michael Addition Explains the Anti-SARS-CoV-2 and Bioactive Properties of Arteannuin B.

Artemisia annua is the plant that produces artemisinin, an endoperoxide-containing sesquiterpenoid used for the treatment of malaria. A. annua extracts, which contain other bioactive compounds, have been used to treat other diseases, including cancer and COVID-19, the disease caused by the virus SARS-CoV-2. In this study, a methyl ester derivative of arteannuin B was isolated when A. annua leaves were extracted with a 1:1 mixture of methanol and dichloromethane. This methyl ester was thought to be formed from the reaction between arteannuin B and the extracting solvent, which was supported by the fact that arteannuin B underwent 1,2-addition when it was dissolved in deuteromethanol. In contrast, in the presence of N-acetylcysteine methyl ester, a 1,4-addition (thiol-Michael reaction) occurred. Arteannuin B hindered the activity of the SARS CoV-2 main protease (nonstructural protein 5, NSP5), a cysteine protease, through time-dependent inhibition. The active site cysteine residue of NSP5 (cysteine-145) formed a covalent bond with arteannuin B as determined by mass spectrometry. In order to determine whether cysteine adduction by arteannuin B can inhibit the development of cancer cells, similar experiments were performed with caspase-8, the cysteine protease enzyme overexpressed in glioblastoma. Time-dependent inhibition and cysteine adduction assays suggested arteannuin B inhibits caspase-8 and adducts to the active site cysteine residue (cysteine-360), respectively. Overall, these results enhance our understanding of how A. annua possesses antiviral and cytotoxic activities.

Synthesis of menarandroside A from dehydroepiandrosterone.

Menarandroside A, which bears a 12α-hydroxypregnenolone steroid backbone, was isolated from the plant, Cynanchum menarandrense. Treatment of extracts from this plant containing menarandroside A against secretin tumor cell line (STC-1) intestinal cells, resulted in an increased secretion of glucagon-like peptide 1 (GLP-1), a peptide that plays a role in the regulation of blood sugar levels. Increase in GLP-1 is beneficial for the treatment of type 2 diabetes. We disclose the synthesis of menarandroside A from dehydroepiandrosterone (DHEA). Key features of this synthesis include: (i) Wittig reaction of the C17-ketone of a 12-oxygenated DHEA derivative to introduce the C17-acetyl moiety, and (ii) the stereoselective reduction of a C12-keto intermediate bearing an sp2-center at C17 to yield the C12α-hydroxy group. In addition, an oxidation of a methyl enol ether derivative to an α-hydroxy methyl ester using tetrapropylammonium perruthenate (TPAP) and N-methyl-morpholine-N-oxide (NMO) was discovered.

The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19.

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.

A biosynthetically inspired synthesis of (-)-berkelic acid and analogs.

We describe a complete account of our total synthesis and biological evaluation of (-)-berkelic acid and analogs. We delineate a synthetic strategy inspired by a potentially biomimetic union between the natural products spicifernin and pulvilloric acid. After defining optimal parameters, we executed a one-pot silver-mediated in situ dehydration of an isochroman lactol to methyl pulvillorate, the cycloisomerization of a spicifernin-like alkynol to the corresponding exocyclic enol ether, and a subsequent cycloaddition to deliver the tetracyclic core of berkelic acid. Our studies confirm that the original assigned berkelic acid structure is not stable and equilibrates into a mixture of 4 diastereomers, fully characterized by X-ray crystallography. In addition to berkelic acid, C22-epi-berkelic acid, and nor-berkelic acids, we synthesized C26-oxoberkelic acid analogs that were evaluated against human cancer cell lines. In contrast to data reported for natural berkelic acid, our synthetic material and analogs were found to be devoid of activity.

Formation and Cleavage of C-C Bonds by Enzymatic Oxidation-Reduction Reactions.

Many oxidation-reduction (redox) enzymes, particularly oxygenases, have roles in reactions that make and break C-C bonds. The list includes cytochrome P450 and other heme-based monooxygenases, heme-based dioxygenases, nonheme iron mono- and dioxygenases, flavoproteins, radical S-adenosylmethionine enzymes, copper enzymes, and peroxidases. Reactions involve steroids, intermediary metabolism, secondary natural products, drugs, and industrial and agricultural chemicals. Many C-C bonds are formed via either (i) coupling of diradicals or (ii) generation of unstable products that rearrange. C-C cleavage reactions involve several themes: (i) rearrangement of unstable oxidized products produced by the enzymes, (ii) oxidation and collapse of radicals or cations via rearrangement, (iii) oxygenation to yield products that are readily hydrolyzed by other enzymes, and (iv) activation of O2 in systems in which the binding of a substrate facilitates O2 activation. Many of the enzymes involve metals, but of these, iron is clearly predominant.

Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO(3+), for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1.

The enzyme cytochrome P450 11A1 cleaves the C20-C22 carbon-carbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carbon-carbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound I (FeO(3+)). Compound I can either (i) abstract an O-H hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol. The mechanism of this carbon-carbon bond cleavage step was tested using (18)O-labeled molecular oxygen and purified P450 11A1. P450 11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of molecular oxygen ((18)O2), and coupled assays were used to trap the labile (18)O atoms in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting products were derivatized and the (18)O content was analyzed by high-resolution mass spectrometry. P450 11A1 showed no incorporation of an (18)O atom into either of its carbon-carbon bond cleavage products, pregnenolone and isocaproaldehyde . The positive control experiments established retention of the carbonyl oxygens in the enzymatic products during the trapping and derivatization processes. These results reveal a mechanism involving an electrophilic Compound I species that reacts with nucleophilic hydroxy groups in the 20R,22R-dihydroxycholesterol intermediate of the P450 11A1 reaction to produce the key steroid pregnenolone.

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1: 18O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN.

Cytochrome P450 (P450) reactions can involve C-C bond cleavage, and several of these are critical in steroid and sterol biosynthesis. The mechanisms of P450s 11A1, 17A1, 19A1, and 51A1 have been controversial, in the context of the role of ferric peroxide (FeO2 (-)) versus perferryl (FeO(3+), compound I) chemistry. We reinvestigated the 17α-hydroxyprogesterone and 17α-hydroxypregnenolone 17α,20-lyase reactions of human P450 17A1 and found incorporation of one (18)O atom (from (18)O2) into acetic acid, consonant with proposals for a ferric peroxide mechanism (Akhtar, M., Lee-Robichaud, P., Akhtar, M. E., and Wright, J. N. (1997) J. Steroid Biochem. Mol. Biol. 61, 127-132; Akhtar, M., Wright, J. N., and Lee-Robichaud, P. (2011) J. Steroid Biochem. Mol. Biol. 125, 2-12). However, the reactions were supported by iodosylbenzene (a precursor of the FeO(3+) species) but not by H2O2 We propose three mechanisms that can involve the FeO(3+) entity and that explain the (18)O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17α-hydroxylated products to yield 16,17α-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6ß-hydroxylation of 16α,17α-dihydroxyprogesterone and the oxidation of both 16α,17α-dihydroxyprogesterone and 16α,17α-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17α,20-lyase reaction cannot be excluded.

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase: STRUCTURE OF THE ENZYME·PROGESTERONE SUBSTRATE COMPLEX AND RATE-LIMITING C-H BOND CLEAVAGE.

Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 10(7) M(-1) s(-1)) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 10(7) M(-1) s(-1)) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.

The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1).

The steroid hydroxylation and carbon-carbon bond cleavage activities of cytochrome P450 17A1 (CYP17A1) are responsible for the production of glucocorticoids and androgens, respectively. The inhibition of androgen synthesis is an important strategy to treat androgen-dependent prostate cancer. We discuss the different enzymatic activities towards the various substrates of CYP17A1, demonstrating its promiscuity. Additionally, a novel interhelical interaction is proposed between the F-G loop and the B'-helix to explain the 16α-hydroxylase activity of human CYP17A1 with progesterone as the substrate. The techniques used by biochemists to study this important enzyme are also summarized. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'.

Mechanism of the third oxidative step in the conversion of androgens to estrogens by cytochrome P450 19A1 steroid aromatase.

Aromatase is the cytochrome P450 enzyme that cleaves the C10-C19 carbon-carbon bond of androgens to form estrogens, in a three-step process. Compound I (FeO(3+)) and ferric peroxide (FeO2(-)) have both been proposed in the literature as the active iron species in the third step, yielding an estrogen and formic acid. Incubation of purified aromatase with its 19-deutero-19-oxo androgen substrate was performed in the presence of (18)O2, and the products were derivatized using a novel diazo reagent. Analysis of the products by high-resolution mass spectrometry showed a lack of (18)O incorporation in the product formic acid, supporting only the Compound I pathway. Furthermore, a new androgen 19-carboxylic acid product was identified. The rates of nonenzymatic hydration of the 19-oxo androgen and dehydration of the 19,19-gem-diol were shown to be catalytically competent. Thus, the evidence supports Compound I and not ferric peroxide as the active iron species in the third step of the steroid aromatase reaction.

A-ring modified steroidal azoles retaining similar potent and slowly reversible CYP17A1 inhibition as abiraterone.

Abiraterone acetate is a potent inhibitor of human cytochrome P450c17 (CYP17A1, 17α-hydroxylase/17,20-lyase) and is clinically used in combination with prednisone for the treatment of castration-resistant prostate cancer. Although many studies have documented the potency of abiraterone (Abi) in a variety of in vitro and in vivo systems for several species, the exact potency of Abi for human CYP17A1 enzyme has not yet been determined, and the structural requirements for high-potency steroidal azole inhibitors are not established. We synthesized 4 Abi analogs differing in the A-B ring substitution patterns: 3α-hydroxy-Δ(4)-Abi (13), 3-keto-Δ(4)-Abi (11), 3-keto-5α-Abi (6), and 3α-hydroxy-5α-Abi (5). We measured the spectral binding constants (Ks) using purified and modified human CYP17A1 along with the determination constants (Ki) applying a native human CYP17A1 enzyme in yeast microsomes for these compounds as well as for ketoconazole. For Abi, 3-keto-Δ(4)-Abi, 3-keto-5α-Abi, and 3α-hydroxy-5α-Abi, the type 2 spectral changes gave the best fit for a quadratic equation, since in these experiments Ks values were 0.1-2.6nM, much lower than that for ketoconazole and 3α-hydroxy-Δ(4)-Abi (Ks values were 140 and 1660nM, respectively). Inhibition experiments showed mixed inhibition patterns with Ki values of 7-80nM. Abi dissociation from the CYP17A1-Abi complex was incomplete and slow; the t1/2 for dissociation was 1.8h, with 55% of complex remaining after 5h. We conclude that Abi and the 3 related steroidal azoles (3-keto-Δ(4)-Abi, 3-keto-5α-Abi, and 3α-hydroxy-5α-Abi), which also mimic natural substrates, are extraordinarily potent inhibitors of human CYP17A1, whereas the 3α-hydroxy-Δ(4)-Abi is moderately potent and comparable to ketoconazole.