Similar Effect of Rifampin and Other Rifamycin Derivatives on Vaccinia Virus Morphogenesis

Membrane-limited structures, resembling virus envelope precursors previously shown to form during the interruption of poxvirus assembly by rifampin, were now observed by electron microscopy in vaccinia-infected HeLa cells treated with a series of rifamycin derivatives. The active compounds N-demethyl rifampin, AF/DMI, and 3-formyl rifamycin SV lacked, respectively, a methyl group, the piperazine ring, and the hydrazone portion of rifampin. A vaccinia mutant selected only for resistance to rifampin was also resistant to the effect on morphogenesis produced by all of the rifamycin derivatives. We concluded that this antiviral effect was specific and was a property associated with the macrocyclic ring rather than the hydrazone-containing side chain of rifampin. In addition to their effects on vaccinia morphogenesis, 3-formyl rifamycin SV and AF/DMI had unusual cytotoxic effects.     

Betulinic acid derivatives as HIV-1 antivirals

Betulinic acid (BA) derivatives are low molecular weight organic compounds synthesized from a plant-derived natural product. Several BA derivatives are potent and highly selective inhibitors of HIV-1. Depending on the specific side-chain modification, these compounds function by inhibiting HIV fusion or, as recently demonstrated, by interfering with a specific step in HIV-1 maturation. BA derivatives have potential as novel HIV-1 therapies, and additional studies of their mechanisms of action are likely to further define the novel targets of these compounds and elucidate the basic biology of HIV-1 fusion and maturation. In this review, recent studies of the novel mechanisms of action of this interesting class of antiviral compounds are discussed.     

Rifamycins: an insight into biological activity based on structural investigations

The structures of two compounds of the antibiotic rifamycin series have been studied by X-ray methods: rifamycin B, C₃₉H₄₉NO₁₄, which is active on bacterial RNA polymerase, and rifamycin Y, C₃₉H₄₇NO₁₅, which is devoid of any antibacterial activity. In both cases their p-iodoanilide derivative has been used and the final crystal structure data are now given.A comparative study of the structures of rifamycin B and rifamycin Y, and of the structures of two other ansa-compounds with similar activity on bacterial RNA polymerase, tolypomycin Y and streptovaricin C, together with the analysis of the activity ratios of a series of semisynthetic rifamycins, makes it possible to suggest that the following constitutional and conformational features are common to all the ansa-compounds with anti-bacterial activity: (i) a naphthoquinone or naphthohydroquinone nucleus with free oxygen functions, O(1) and O(2), on C(1) and C(8) respectively: (ii) an amide nitrogen attached to C(2); (iii) a 17-membered chain bridging the chromophoric group; (iv) two free hydroxyl groups attached to atoms C(21) and C(23) of the ansa-bridge and in the same relative positions with respect to each other and with respect to O(1) and O(2). However, the constitution and the conformation of the remaining part of the ansa-bridge can be rather different in different compounds.     

Rifamycin derivatives: specific inhibitors of nucleic acid polymerases

Rifampicin and three rifamycin SV derivatives with different lipophilic side chains were tested as inhibitors of a number of purified enzymes including the α and αβ forms of RNA-directed DNA polymerase of avian myeloblastosis virus (AMV). $\text{AF}\text{ABDMP}$ (2,5-dimethyl-4-N-benzyl demethyl rifampicin), $\text{AF}\text{013}$ (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 (rifamycin SV with a dicyclohexylalkyl substituted piperidyl ring at the 3-position) at concentrations less than 20 to 40 μg/ml completely inhibited the RNA- and DNA-directed DNA polymerase and RNase H activities of both AMV enzymes. Rifampicin was inactive at 100 μg/ml. When used against a variety of non-polymerizing enzymes such as alkaline phosphatase, glutamate-oxaloacetate transaminase, DNase I, and RNase A, these derivatives were inactive at drug concentrations between 100 and 200 μg/ml. Polynucleotide phosphorylase was inhibited slightly by all three derivatives. These results support the idea that rifamycin SV derivatives with appropriate 3-substituted side-chains are specific inhibitors of nucleic acid polymerizing enzymes.     

Synthesis of phenazine and phenoxazinone derivatives of geldanamycin as potential polymerase inhibitors

Derivatives of geldanamycin—phenazines and phenoxazinones, some of them analogous to rifazine in the rifamycin series—have been synthesized. Most of the geldanamycin derivatives were highly effective inhibitors of RNA-dependent DNA polymerase from Rauscher leukemia virus. A simple analog containing the aromatic chromophore of the derivatives but without the ansa ring also showed viral inhibition.     

Effect of rifamycin and tilorone derivatives on Friend virus leukemia in mice.

Several derivatives of rifamycin, and analogs of the tilorone-fluoranthene group were tested for inhibition of splenic enlargement in Friend virus leukemia. At least three members of the rifamycin group caused significant inhibition (31-49%) as did at least three members of the tilorone group (32-48%). These six compounds are among those found by others (6, 7) to be most inhibitory in vitro to the RNA-directed DNA polymerase of oncornaviruses. However our studies do not furnish direct evidence for or against a role of inhibition of the viral enzyme in the suppression of splenomegaly. None of the agents was as effective as methotrexate, which caused 90-92% inhibition. The activity of five of the agents was reduced, rather than enhanced by the injection of adjuvants (M. butyricum and pertussis vaccine). Three of the agents had a subtractive, rather than an additive effect on the inhibition caused by methotrexate alone.     

Streptovaricins inhibit Focus Formation by MSV (MLV) Complex

We recently reported that the streptovaricins inhibit the reaction by which DNA is transcribed from the RNA template resident in murine leukaemia virions (MLV)¹. The reports^{2, 3} which first indicated that this DNA polymerase is present in oncogenic RNA viruses have been confirmed and extended^4–8. The enzyme provides a mechanism whereby an RNA virus can insert stable genetic information into a host chromosome. Gallo and co-workers described an RNA dependent DNA polymerase in lymphoblastic leukaemic cells which was inhibited by N-demethylrifampicin⁹ and this antibiotic, together with a number of other rifamycin derivatives, also inhibited the oncogenic viral DNA polymerase¹⁰. Like the streptovaricins, the rifamycins are ansa macrolide antibiotics (Fig. 1).     

Comparative study of the effect of different rifamycins on bacterial cell metabolism and on the RNA-polymerase reaction in a cell-free system]

The results of the study on the inhibitory effect of a number of rifamycin derivatives, such as rifamycin B, rifamycin O, rifamycin, rifamycin A, 25-desacetylrifampicin and rifampicin are presented. It was shown that rifampicin had the highest inhibitory effect on the synthesis of RNA in the cells of E. coli and Staph. aureus. It inhibited the above process by 93.0 and 98.8 per cent respectively. The data on the cells of Staph. aureus, as well as the data on comparison of the inhibitory effect of rifampicin derivatives with respect to the RNA-polymerase reaction in acellular systems are presented.     

Synthesis and antiviral activity of aryl phosphoramidate derivatives of beta-D- and beta-L-C-5-substituted 2',3'-didehydro-2',3'-dideoxy-uridine bearing linker arms

We have previously reported the synthesis and evaluation of potent anti-human immunodeficiency virus compounds based on beta-D-d4T analogues bearing a tether attached at the C-5 position and their beta-L-counterparts. Initial study revealed a requirement for an alkyl side-chain with an optimal length of 12 carbons for a weak antiviral activity. As a continuation of that work, we have now prepared the corresponding phosphoramidate derivatives as possible membrane-permeable prodrugs. Phosphorochloridate chemistry gave the target phosphoramidates which were tested for anti-human immunodeficiency virus type 1 activity; unfortunately, they were devoid of anti-HIV activity.     

Synthesis and antiviral activity of aryl phosphoramidate derivatives of β-D- and β-L-C-5-Substituted 2’,3’-didehydro-2’,3’-dideoxy-uridine bearing linker arms

We have previously reported the synthesis and evaluation of potent anti-human immunodeficiency virus compounds based on β-D-d4T analogues bearing a tether attached at the C-5 position and their β-L-counterparts. Initial study revealed a requirement for an alkyl side-chain with an optimal length of 12 carbons for a weak antiviral activity. As a continuation of that work, we have now prepared the corresponding phosphoramidate derivatives as possible membrane-permeable prodrugs. Phosphorochloridate chemistry gave the target phosphoramidates which were tested for anti-human immunodeficiency virus type 1 activity; unfortunately, they were devoid of anti-HIV activity.     

Chemical Derivatives of a Small Molecule Deubiquitinase Inhibitor Have Antiviral Activity against Several RNA Viruses

Most antiviral treatment options target the invading pathogen and unavoidably encounter loss of efficacy as the pathogen mutates to overcome replication restrictions. A good strategy for circumventing drug resistance, or for pathogens without treatment options, is to target host cell proteins that are utilized by viruses during infection. The small molecule WP1130 is a selective deubiquitinase inhibitor shown previously to successfully reduce replication of noroviruses and some other RNA viruses. In this study, we screened a library of 31 small molecule derivatives of WP1130 to identify compounds that retained the broad-spectrum antiviral activity of the parent compound in vitro but exhibited improved drug-like properties, particularly increased aqueous solubility. Seventeen compounds significantly reduced murine norovirus infection in murine macrophage RAW 264.7 cells, with four causing decreases in viral titers that were similar or slightly better than WP1130 (1.9 to 2.6 log scale). Antiviral activity was observed following pre-treatment and up to 1 hour postinfection in RAW 264.7 cells as well as in primary bone marrow-derived macrophages. Treatment of the human norovirus replicon system cell line with the same four compounds also decreased levels of Norwalk virus RNA. No significant cytotoxicity was observed at the working concentration of 5 µM for all compounds tested. In addition, the WP1130 derivatives maintained their broad-spectrum antiviral activity against other RNA viruses, Sindbis virus, LaCrosse virus, encephalomyocarditis virus, and Tulane virus. Thus, altering structural characteristics of WP1130 can maintain effective broad-spectrum antiviral activity while increasing aqueous solubility.     

Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and binding to tear lipocalin

A mutant of Mycobacterium smegmatis is a potential class I model substitute for Mycobacterium tuberculosis. Because not all of the rifamycins have been tested in this organism, we determined bactericidal profiles for the 6 major rifamycin derivatives. The profiles closely mirrored those established for M. tuberculosis. Rifalazil was confirmed to be the most potent rifamycin. Because the tuberculous granuloma presents a harshly oxidizing environment we explored the effects of oxidation on rifamycins. Mass spectrometry confirmed that three of the six major rifamycins showed autoxidation in the presence of trace metals. Oxidation could be monitored by distinctive changes including isosbestic points in the ultraviolet–visible spectrum. Oxidation of rifamycins abrogated anti-mycobacterial activity in M. smegmatis. Protection from autoxidation was conferred by binding susceptible rifamycins to tear lipocalin, a promiscuous lipophilic protein. Rifalazil was not susceptible to autoxidation but was insoluble in aqueous solution. Solubility was enhanced when complexed to tear lipocalin and was accompanied by a spectral red shift. The positive solvatochromism was consistent with robust molecular interaction and binding. Other rifamycins also formed a complex with lipocalin, albeit to a lesser extent. Protection from oxidation and enhancement of solubility with protein binding may have implications for delivery of select rifamycin derivatives.     

A solvent effect on the side-chain conformation of phenylalanine derivatives and phenylalanine residuces in dipeptides

Three phenylalanine derivatives, Ac-Phe-NHMe, H-Phe-NHMe, and Ac-Phe-OH, were selected as models of Phe residues situated at the internal, the N-terminal, and the C-terminal positions of peptide chains, respctively. The side-chain conformations of the three compounds were analyzed from the vicnal coupling constants ³J_αβR and ³J_αβS, of their ¹H- nmr spectra measured in various organic sovlent. The two β-protons were unambiguously assined by use of sterospecifically β-monodeuterated phenylalanines. The pro-S β-proton was always situated at lower field than the pro-R one when they were observed separately. The results of a solvent effect on the conformation of the tree compounds demonstrated that the rotamer populations are remarkable sensitive of the three compounds demonstrated that the rotamer populations are remarkably sensitive to solvent polarity and that the tendencies of the solvent effects are quite different from each other. Ac-Phe-OH Showed a trend similar to that of Ac-Phe-OEt reported by early workers. The rotamer populations of other derivatives (Ac-Phe-NMe₂, Ac-Phe-NH₂, Ac-Phe-OBu^t, and Ac-Phe-OBzl) and of Phe residues in some N-acetyl dipeptde esters (Ac-Phe-Gly-OMe, Ac-Phe-Val-OMe, and Ac-Gly-Phe-OMe) were also examined in several sovent, and it was found that substituents of the Phe carboxyl group—amides or esters—determine the tendency of the solvent effect. These results are interesting in the side-chain conformations of Phe residues in peptides and proteins in an environment of low polarity can be disscussed on this experimental basis. Factors responsible for the solvent effect are discussed from (1) a structural comparison of the compunds with various carboxylic substituents, (2) an expriment with cyclohexylalanine derivatives, and (3) the measurement in mixed solvents wiht similar polarity.