Peter K. Chiang

Biological effects of inhibitors of S-adenosylhomocysteine hydrolase

S-Adenosylhomocysteine (AdoHcy), formed after the donation of the methyl group of S-adenosylmethionine to a methyl acceptor, is hydrolyzed to adenosine and homocysteine by AdoHcy hydrolase physiologically. The administration of the inhibitors of AdoHcy hydrolase to cells or animals normally results in an accumulation of cellular AdoHcy higher than those found in controls, which is often accompanied by a simultaneous rise in S-adenosylmethionine because of the feedback inhibition by AdoHcy on most methylation reactions. AdoHcy hydrolase has become a tantalizing pharmacological target for inhibition since its blockade can affect cellular methylation of phospholipids, proteins, small molecules, DNA, and RNA. Indeed, all of these different methylation reactions have been found to be inhibitable by the nucleoside inhibitors/substrates of AdoHcy hydrolase. Among the interesting effects are the activation of genes, induction of cellular differentiation, increased expression of transcription factors, and sometimes the repression of genes. Furthermore, some of the nucleosides show remarkable antiviral activities in vitro and in vivo. However, the mode of action of the inhibitors appears complex. Although the inhibition of methylation might account for some of the biological effects, the ability of some of the nucleoside inhibitors to undergo metabolic phosphorylation to nucleotides may account for part of their biological activities. The defining mode of action responsible for their biological effects still awaits biochemical elaboration, especially regarding their antiviral effects, induction of genes, or cellular differentiation.

Perturbation of biochemical transmethylations by 3-deazaadenosine in vivo

Elevated levels of adenosylmethionine (AdoMet) and adenosylhomocysteine (AdoHcy) were observed in livers of rats injected with 3-deazaadenosine, an inhibitor of adenosylhomocysteine hydrolase. A marked appearance of 3-deazaadenosylhomocysteine was observed. The ratio of AdoMet/AdoHcy in the liver was reduced from a normal value of 4.5 to 1.6 after two injections, and to 1.3 after three injections. Perturbation of biochemical transmethylations was achieved in rats injected with 3-deazaadenosine, as evidenced by: (a) a reduction in the level of creatine in the liver; (b) a decrease in the urinary excretion of 3-methoxy-4-hydroxymandelic acid; and (c) a drastic reduction in the methylation of lipids, as measured by methyl-3H-incorporation from [methyl-3H]AdoMet. These observations support the hypothesis that the ratio AdoMet/AdoHcy may be of critical importance in the regulation of biological methylations. The enzyme, adenosylhomocysteine hydrolase, therefore, may be considered a prime biological target in terms of in vivo regulation of transmethylation reactions.

3-Deazaneplanocin: A new and potent inhibitor of S-adenosylhomocysteine hydrolase and its effects on human promyelocytic leukemia cell line HL-60

3-Deazaneplanocin, a new carbocyclic analog of adenosine, was synthesized as an inhibitor of S-adenosylhomocysteine hydrolase. The Ki of 3-deazaneplanocin for a purified hamster liver preparation of S-adenosylhomocysteine hydrolase was 5 X 10(-11) M, making this inhibitor 250-fold more potent than the previously known most potent inhibitor of this enzyme, 3-deazaaristeromycin. Inhibition was competitive with the substrate adenosine. Human promyelocytic leukemia (HL-60) cells treated with 10(-5) M 3-deazaneplanocin showed a pronounced elevation in S-adenosylhomocysteine which was 4-fold greater than that produced by an equimolar concentration of 3-deazaaristeromycim. This effect preceded a moderate reduction in cell growth and viability following continuous exposure for 6 days. Cellular differentiation as monitored by the reduction of nitroblue tetrazolium was not markedly affected except after 4 days exposure to 10(-5) M 3-deazaneplanocin where 60% of the viable cells were positive. These results indicate that 3-deazaneplanocin may have therapeutic potential as an anticancer or antiviral drug.

Discovery of Trypanocidal Compounds by Whole Cell HTS of Trypanosoma brucei

Chemotherapy against human African trypanosomiasis relies on four drugs that cause frequent and occasionally severe side‐effects. Because human African trypanosomiasis is a disease of poor people in Africa, the traditional market‐driven pathways to drug development are not available. One potentially rapid and cost‐effective approach to identifying and developing new trypanocidal drugs would be high throughput‐screening of existing drugs already approved for other uses, as well as clinical candidates in late development. We have developed an ATP‐bioluminescence assay that could be used to rapidly and efficiently screen compound libraries against trypanosomes in a high throughput‐screening format to validate this notion. We screened a collection of 2160 FDA‐approved drugs, bioactive compounds and natural products to identify hits that were cytotoxic to cultured Trypanosoma brucei at a concentration of 1 μm or less. This meant that any hit identified would be effective at a concentration readily achievable by standard drug dosing in humans. From the screen, 35 hits from seven different drug categories were identified. These included the two approved trypanocidal drugs, suramin and pentamidine, several other drugs suspected but never validated as trypanocidal, and 17 novel trypanocidal drugs.

Angiogenesis inhibitors specific for methionine aminopeptidase 2 as drugs for malaria and leishmaniasis.

Methionine aminopeptidase 2 (MetAP2) is responsible for the hydrolysis of the initiator methionine molecule from the majority of newly synthesized proteins. We have cloned the MetAP2 gene from the mal

3-Deazaadenosine, an inhibitor of adenosylhomocysteine hydrolase, inhibits reproduction of rous sarcoma virus and transformation of chick embryo cells

Abstract 3-Deazaadenosine, a known potent inhibitor of adenosylhomocysteine hydrolase, exerts an inhibitory effect on the reproduction of Rous sarcoma virus (RSV-BH) and on the malignant transformation of chick embryo cells by this virus. The inhibitory effect is reversible. The increased capacity for glucose uptake, a biochemical characteristic of chick embryo cells transformed by RSV-BH, is inhibited by 3-deazaadenosine, and the ability of the infected cells to grow in suspension is blocked. After prolonged exposure to 3-deazaadenosine, the morphological phenotype characteristic of transformed cells largely disappeared, and transformed cells resembled noninfected cells. Deazaadenosine inhibits the reproduction of Sindbis virus, Newcastle disease virus, and vesicular stomatitis virus to a lesser degree than RSV-BH. Deazaadenosine, 0.1 m M , has no effect on DNA or protein synthesis in cells, and only a slight effect on RNA synthesis. No incorporation of 3-deaza[ 14 C]adenosine into cellular nucleic acids was found. Deazaadenosine produces an increase in the intracellular level of adenosylhomocysteine, with the concomitant appearance of a relatively large amount of 3-deazaadenosylhomocysteine; the ratio of intracellular adenosylmethionine to adenosylhomocysteine, or (adenosylhomocysteine + 3-deazaadenosylhomocysteine) is decreased from 150 to 19, and 1.4 respectively. It is postulated that 3-deazaadenosine inhibits virus activities by its ability to inhibit adenosylhomocysteine hydrolase, resulting in an inhibition of methylation reaction(s) required for virus growth and replication.

Synthesis and Anti-Breast Cancer Activities of Substituted Quinolines

Promising anti-breast cancer agents derived from substituted quinolines were discovered. The quinolines were readily synthesized in a large scale from a sequence of reactions starting from 4-acetamidoanisole. The Michael addition product was isolated as the reaction intermediate in the ring closing reaction of 4-amino-5-nitro-2-(3-trifluoromethylphenyloxy)anisole with methyl vinyl ketone leading to 6-methoxy-4-methyl-8-nitro-5-(3-trifluoromethylphenyloxy)quinoline (14). The amino function of 8-amino-6-methoxy-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, prepared from 14, was connected to various side chains via alkylation with N-(3-iodopropyl)phthalimide, Michael addition with acrylonitrile, and reductive amination with various heterocycle carboxaldehydes, such as imidazole-4-carboxaldehyde, thiophene-2-carboxaldehyde, and 2-furaldehyde. Effects of the substituted quinolines on cell viability of T47D breast cancer cells using trypan blue exclusion assay were examined. The results showed that the IC(50) value of 6-methoxy-8-[(2-furanylmethyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline is 16+/-3nM, the lowest IC(50) out of all the quinolines tested. IC(50) values of three other quinolines are in the nanomolar range, a desirable range for pharmacological testing.

Irreversible inhibition of S-adenosylhomocysteine hydrolase by nucleoside analogs.

Abstract A large number of nucleoside analogs have been found to inactivate S -adenosylhomocysteine (AdoHcy) hydrolase in a time-dependent irreversible manner. There are two classes of these irreversible inhibitors: (A) analogs that inactivate the enzyme in a pseudofirst-order process and are devoid of any side chain at the 5′-OH group; (B) analogs that inactivate the enzyme in a time-dependent but curvilinear process, and generally have a side chain at the 5′ position. Among the more potent irreversible inhibitors are 2-chloroadenosine, 9-β- d -arabinofuranosyladenine (Ara-A), and (±)aristeromycin. Release of adenine base from adenosine or Ara-A in the presence of AdoHcy hydrolase was observed, thus supporting the proposed catalytic mechanism of AdoHcy hydrolase, that entails the transient formation of 3′-ketoadenosine during enzymatic catalysis of either the formation or hydrolysis of AdoHcy. Both Ara-A and adenosine may exert their irreversible inactivation by a suicide mechanism, but nucleosides such as 5′-iodo-5′-deoxyadenosine and 3′-deoxyadenosine are probably strictly irreversible inhibitors per se in view of the catalytic mechanism proposed for AdoHcy hydrolase. Labeling of AdoHcy hydrolase, perhaps covalent in nature, by radioactive Ara-A and adenosine was demonstrated by gel electrophoresis.

Plasmodium falciparum: antimalarial activity in culture of sinefungin and other methylation inhibitors.

Abstract A culture line of Plasmodium falciparum (FCR-3/Gambia) was exposed in vitro for a 2-day period to several analogs of adenosylhomocysteine. Minimal concentrations giving complete inhibition of growth were 0.2 m M for 3-deazaadenosine, 0.2 m M for 5′-deoxy-5′-(isobutylthio)-3-deazaadenosine, and 0.3 μM for sinefungin. The effects of the first two of these compounds were potentiated by homocysteine-thiolactone, suggesting that they were inhibiting methylation reaction(s) indirectly via adenosylhomocysteine hydrolase (EC 3.3.1.1).

Hydroxychloroquine in human breast milk

SummaryHydroxychloroquine 3.2 µg was detected in breast milk from a woman given 800 mg over 48 hour.