Benzoylphenyl thiocyanates are new, effective inhibitors of the mycobacterial resuscitation promoting factor B protein.

BACKGROUND: Resuscitation promoting factors (Rpfs) are the proteins involved in the process of reactivation of the dormant cells of mycobacteria. Recently a new class of nitrophenylthiocyanates (NPTs), capable of inhibiting the biological and enzymatic activities of Rpfs has been discovered. In the current study the inhibitory properties of the compounds containing both nitro and thiocyanate groups alongside with the compounds with the modified number and different spatial location of the substituents are compared. METHODS: New benzoylphenyl thiocyanates alongside with nitrophenylthiocyanates were tested in the enzymatic assay of bacterial peptidoglycan hydrolysis as well as against strains of several actinobacteria (Mycobacterium smegmatis, Mycobacterium tuberculosis) on in-lab developed models of resuscitation of the dormant forms. RESULTS: Introduction of the additional nitro and thiocyanate groups to the benzophenone scaffold did not influence the inhibitory activity of the compounds. Removal of the nitro groups analogously did not impair the functional properties of the molecules. Among the tested compounds two molecules without nitro group: 3-benzoylphenyl thiocyanate and 4-benzoylphenyl thiocyanate demonstrated the maximum activity in both enzymatic assay (inhibition of the Rpf-mediated peptidoglycan hydrolysis) and in the resuscitation assay of the dormant M. tuberculosis cells. CONCLUSIONS: The current study demonstrates dispensability of the nitro group in the NPT's structure for inhibition of the enzymatic and biological activities of the Rpf protein molecules. These findings provide new prospects in anti-TB drug discovery especially in finding of molecular scaffolds effective for the latent infection treatment.

Molecular mechanism of a specific capsid binder resistance caused by mutations outside the binding pocket.

Enteroviruses cause various acute and chronic diseases. The most promising therapeutics for these infections are capsid-binding molecules. These can act against a broad spectrum of enteroviruses, but emerging resistant virus variants threaten their efficacy. All known enterovirus variants with high-level resistance toward capsid-binding molecules have mutations of residues directly involved in the formation of the hydrophobic binding site. This is a first report of substitutions outside the binding pocket causing this type of drug resistance: I1207K and I1207R of the viral capsid protein 1 of coxsackievirus B3. Both substitutions completely abolish the antiviral activity of pleconaril (a capsid-binding molecule) but do not affect viral replication rates in vitro. Molecular dynamics simulations indicate that the resistance mechanism is mediated by a conformational rearrangement of R1095, which is a neighboring residue of 1207 located at the heel of the binding pocket. These insights provide a basis for the design of resistance-breaking inhibitors.

Pyrazolopyrimidines: Potent Inhibitors Targeting the Capsid of Rhino- and Enteroviruses.

There are currently no drugs available for the treatment of enterovirus (EV)-induced acute and chronic diseases such as the common cold, meningitis, encephalitis, pneumonia, and myocarditis with or without consecutive dilated cardiomyopathy. Here, we report the discovery and characterization of pyrazolopyrimidines, a well-tolerated and potent class of novel EV inhibitors. The compounds inhibit the replication of a broad spectrum of EV in vitro with IC50 values between 0.04 and 0.64 µM for viruses resistant to pleconaril, a known capsid-binding inhibitor, without affecting cytochrome P450 enzyme activity. Using virological and genetics methods, the viral capsid was identified as the target of the most promising, orally bioavailable compound 3-(4-trifluoromethylphenyl)amino-6-phenylpyrazolo[3,4-d]pyrimidine-4-amine (OBR-5-340). Its prophylactic as well as therapeutic application was proved for coxsackievirus B3-induced chronic myocarditis in mice. The favorable pharmacokinetic, toxicological, and pharmacodynamics profile in mice renders OBR-5-340 a highly promising drug candidate, and the regulatory nonclinical program is ongoing.

QSAR analysis of [(biphenyloxy)propyl]isoxazoles: agents against coxsackievirus B3.

BACKGROUND: Antiviral drugs are urgently needed for the treatment of acute and chronic diseases caused by enteroviruses such as coxsackievirus B3 (CVB3). The main goal of this study is quantitative structure-activity relationship (QSAR) analysis of anti-CVB3 activity (clinical CVB3 isolate 97927 [log IC50, µM]) and investigation of the selectivity of 25 ([biphenyloxy]propyl)isoxazoles, followed by computer-aided design and virtual screening of novel active compounds. DISCUSSION: The 2D QSAR obtained models are quite satisfactory (R(2) = 0.84-0.99, Q(2) = 0.76-0.92, R(2)(ext) = 0.62-0.79). Compounds with high antiviral activity and selectivity have to contain 5-trifluoromethyl-[1,2,4]oxadiazole or 2,4-difluorophenyl fragments. Insertion of 2,5-dimethylbenzene, napthyl and especially biphenyl substituents into investigated compounds substantially decreases both their antiviral activity and selectivity. Several compounds were proposed as a result of design and virtual screening. A high level of activity of 2-methoxy-1-phenyl-1H-imidazo[4,5-c]pyridine (sm428) was confirmed experimentally. CONCLUSION: Simplex representation of molecular structure allows successful QSAR analysis of anti-CVB3 activity of ([biphenyloxy]propyl)isoxazole derivatives. Two possible ways of battling CVB3 are considered as a future perspective.

Per aspera ad astra: application of Simplex QSAR approach in antiviral research.

This review explores the application of the Simplex representation of molecular structure (SiRMS) QSAR approach in antiviral research. We provide an introduction to and description of SiRMS, its application in antiviral research and future directions of development of the Simplex approach and the whole QSAR field. In the Simplex approach every molecule is represented as a system of different simplexes (tetratomic fragments with fixed composition, structure, chirality and symmetry). The main advantages of SiRMS are consideration of the different physical-chemical properties of atoms, high adequacy and good interpretability of models obtained and clear procedures for molecular design. The reliability of developed QSAR models as predictive virtual screening tools and their ability to serve as the basis of directed drug design was validated by subsequent synthetic and biological experiments. The SiRMS approach is realized as the complex of the computer program 'HiT QSAR', which is available on request.

New pleconaril and [(biphenyloxy)propyl]isoxazole derivatives with substitutions in the central ring exhibit antiviral activity against pleconaril-resistant coxsackievirus B3.

Amino acid 1092 (AA1092) in capsid protein 1 of coxsackievirus B3 (CVB3) is located in close vicinity to the central phenoxy group of capsid binders (i.e. pleconaril). Whereas isoleucine is associated with drug susceptibility, leucine and methionine confer resistance to pleconaril. In the present study, novel analogues with different substitutions in the central phenoxy group were synthesized to study their influence on anti-CVB3 activity with the aim to overcome pleconaril resistance. Two [(biphenyloxy)propyl]isoxazoles and pleconaril were synthesized without methyl groups in the central phenoxy ring using Suzuki coupling reaction and tested for antiviral activity against the pleconaril-resistant CVB3 Nancy. Furthermore, pleconaril with 3-methyl, 3-methoxy, 3-bromine, 2,3-dimethyl in the central ring as well as the external rings in meta position were synthesized for structure-activity relationship analysis with CVB3 variants containing leucine, methionine or isoleucine at position 1092, other coxsackieviruses B (CVB) as well as several rhinoviruses. The results demonstrate a high impact of substituents in the central ring of capsid inhibitors for anti-enteroviral activity. Pleconaril resistance of CVB3 based on Leu1092 or Met1092 was overcome by unsubstituted analogues or by monosubstitution with 3-methyl as well as 3-bromine in the central phenyl. The 3-bromine derivative inhibited a broad spectrum of CVB and rhinoviruses.

Quantitative structure-activity relationship studies of [(biphenyloxy)propyl]isoxazole derivatives. Inhibitors of human rhinovirus 2 replication.

The 50% cytotoxic concentration (CC50) in HeLa cells, the 50% inhibitory concentration (IC50) against human rhinovirus 2 (HRV-2), and the selectivity index (SI = CC50/IC50) of [(biphenyloxy)propyl]isoxazole derivatives were used to develop quantitative structure-activity relationships (QSAR) based on simplex representation of molecular structure. Statistic characteristics for partial least-squares models are quite satisfactory (R2 = 0.838 - 0.918; Q2 = 0.695 - 0.87) for prediction of CC50, IC50, and SI values and permit the virtual screening and molecular design of new compounds with strong anti-HRV-2 activity. The quality of prognosis for designed compounds was additionally estimated by analysis of domain applicability for each QSAR model. A hypothesis to the effect that terminal benzene substituents must have negative electrostatic potential and definite length (approximately 5.5-5.6 A) to possess strong antiviral activity has been suggested. The quality of developed analysis, i.e., high level of antiviral action of three new designed compounds, has been confirmed experimentally.

Synthesis and antileprosy activity of some dialkyldithiocarbamates.

OBJECTIVES: To investigate the antileprosy potential of a set of original compounds with antimycobacterial activity. METHODS: We developed a facile synthesis of 2-chloro-3-cyano-5-nitropyridine and synthesized a series of 3-cyano-2-dialkyldithiocarbamoyl-5-nitropyridine derivatives. In vivo therapeutic efficacy against Mycobacterium leprae was assessed in the infected mouse footpad model. RESULTS: The compounds were active in vitro against Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium vaccae and Mycobacterium fortuitum, with MICs generally in the range of 0.4-6.25 mg/L. Reduction of the bacterial load in vivo in the mouse footpad and toxic side effects were dependent on the individual structure of the compounds and on the doses applied. Compounds 2a, 3a and 3b reduced the number of M. leprae by two orders of magnitude, comparable to the effect of dapsone. Co-administration of compounds 2a and 3a with dapsone synergistically enhanced the activity. In addition, these compounds were well tolerated over the treatment period of 7.5 months. CONCLUSIONS: Individual synthetic dithiocarbamate derivatives have promising antileprosy activity.

Susceptibility of coxsackievirus B3 laboratory strains and clinical isolates to the capsid function inhibitor pleconaril: antiviral studies with virus chimeras demonstrate the crucial role of amino acid 1092 in treatment.

OBJECTIVES: At present, most promising compounds to treat enterovirus-induced diseases are broad-spectrum capsid function inhibitors which bind into a hydrophobic pocket in viral capsid protein 1 (VP1). Coxsackievirus B3 (CVB3) Nancy was the only prototypic enterovirus strain shown to be pleconaril-resistant. This study was designed to better understand the polymorphism of the hydrophobic pocket in CVB3 laboratory strains and clinical isolates and its implications for treatment with the capsid function inhibitor pleconaril. METHODS: Pleconaril susceptibility was determined in cytopathic effect-inhibitory, plaque reduction or virus yield assays. Sequence analysis of the genome region coding for VP1 and/or subsequent alignment of amino acids lining the hydrophobic pocket of five CVB3 laboratory strains and 20 clinical isolates were carried out. Virus chimeras and computational analysis were used to prove the role of amino acid 1092. RESULTS AND CONCLUSIONS: Despite high conservation of pocket amino acids, polymorphism was detected at positions 1092, 1094 and 1180. Neither Pro-1094-->Thr nor Val-1180-->Ile altered efficacy of pleconaril treatment. But the amino acid at position 1092 was strongly associated with susceptibility of CVB3 to the capsid inhibitor. Whereas leucine was involved in resistance, isoleucine and valine were detected in pleconaril-susceptible CVB3. Results from antiviral assays with hybrid viruses demonstrate the crucial role of amino acid 1092 in pleconaril susceptibility. A resistant cDNA-generated CVB3 became pleconaril-susceptible after accepting parts from the genome region encoding Ile-1092 into its capsid. Computational analysis suggests that conformational changes in the hydrophobic pocket occur when leucine is substituted for isoleucine or valine and that this change leads to susceptibility to pleconaril.

Novel [(biphenyloxy)propyl]isoxazole derivatives for inhibition of human rhinovirus 2 and coxsackievirus B3 replication.

OBJECTIVES: During this study, novel biphenyl derivatives were synthesized and tested for antiviral activity. METHODS: A new method based on the Suzuki coupling reaction has been established for the synthesis of these polysubstituted chain systems. In parallel with cytotoxicity, the antiviral activity of biphenyl derivatives has been determined in cytopathic effect (CPE)-inhibitory assays with the pleconaril-resistant coxsackievirus B3 (CVB3) strain Nancy, human rhinovirus 2 (HRV-2) and 14 (HRV-14) and in plaque reduction assays with the pleconaril-sensitive human isolate CVB3 97-927 in HeLa cells. Based on the results from these investigations the selectivity index (SI) was determined as the ratio of the 50% cytotoxic concentration to the 50% inhibitory concentration. RESULTS: The new method based on the Suzuki coupling reaction includes the condensation of 2,6-dimethyl-4-bromophenol with pentyne chloride by means of potassium carbonate and potassium iodide in N-methylpyrrolidone-2 and yields 5-bromo-1,3-dimethyl-2-(4-pentynyloxy)benzene. Its condensation with methylacetaldoxime results in 3-methylisoxazole derivatives. The following reaction with different benzeneboronic acids by means of tetrakis(triphenylphosphine)-palladium(0) finally yields the corresponding derivatives. Several of the novel synthesized derivatives demonstrated a good antiviral activity on CVB3 (SI > 2 to > 37.5) and a strong anti-HRV-2 activity (SI > 50 to > 200). In contrast, none of the compounds inhibited the HRV-14-induced CPE. CONCLUSIONS: These results indicate that [(biphenyloxy)propyl]isoxazole derivatives are potential inhibitors of HRV-2 and CVB3 replication, and make them promising agents for the specific treatment of these virus infections.

Anti-coxsackievirus B3 activity of 2-amino-3-nitropyrazolo[1,5-a]pyrimidines and their analogs.

A novel class of 2-amino-4-nitropyrazolo[1,5-a]pyrimidines has been identified as potent inhibitors of coxsackievirus B3 replication. The synthesis of these compounds is based on the regioselective reaction of 3,5-diamino-5-nitropyrazole with unsymmetrical beta-diketones at catalysis by hydrochloric acid leading to 2-amino-4-nitropyrazolo[1,5-a]pyrimidines as key steps.