Design of inhibitors of thymidylate kinase from Variola virus as new selective drugs against smallpox: part II.

Acknowledging the importance of studies toward the development of measures against terrorism and bioterrorism, this study aims to contribute to the design of new prototypes of potential drugs against smallpox. Based on a former study, nine synthetic feasible prototypes of selective inhibitors for thymidylate kinase from Variola virus (VarTMPK) were designed and submitted to molecular docking, molecular dynamics simulations and binding energy calculations. The compounds are simplifications of two more complex scaffolds, with a guanine connected to an amide or alcohol through a spacer containing ether and/or amide groups, formerly suggested as promising for the design of selective inhibitors of VarTMPK. Our study showed that, despite the structural simplifications, the compounds presented effective energy values in interactions with VarTMPK and HssTMPK and that the guanine could be replaced by a simpler imidazole ring linked to a -NH2 group, without compromising the affinity for VarTMPK. It was also observed that a positive charge in the imidazole ring is important for the selectivity toward VarTMPK and that an amide group in the spacer does not contribute to selectivity. Finally, prototype 3 was pointed as the most promising to be synthesized and experimentally evaluated. Communicated by Ramaswamy H. Sarma.

Design of inhibitors of thymidylate kinase from Variola virus as new selective drugs against smallpox.

Recently we constructed a homology model of the enzyme thymidylate kinase from Variola virus (VarTMPK) and proposed it as a new target to the drug design against smallpox. In the present work, we used the antivirals cidofovir and acyclovir as reference compounds to choose eleven compounds as leads to the drug design of inhibitors for VarTMPK. Docking and molecular dynamics (MD) studies of the interactions of these compounds inside VarTMPK and human TMPK (HssTMPK) suggest that they compete for the binding region of the substrate and were used to propose the structures of ten new inhibitors for VarTMPK. Further docking and MD simulations of these compounds, inside VarTMPK and HssTMPK, suggest that nine among ten are potential selective inhibitors of VarTMPK.

Variola type IB DNA topoisomerase: DNA binding and supercoil unwinding using engineered DNA minicircles.

Type IB topoisomerases unwind positive and negative DNA supercoils and play a key role in removing supercoils that would otherwise accumulate at replication and transcription forks. An interesting question is whether topoisomerase activity is regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The type IB enzyme from variola virus (vTopo) has proven to be useful in addressing mechanistic questions about topoisomerase function because it forms a reversible 3'-phosphotyrosyl adduct with the DNA backbone at a specific target sequence (5'-CCCTT-3') from which DNA unwinding can proceed. We have synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site that provides highly defined substrates for exploring the effects of supercoil density on DNA binding, strand cleavage and ligation, and unwinding. We observed no topological dependence for binding of vTopo to these supercoiled MC DNAs, indicating that affinity-based targeting to supercoiled DNA regions by vTopo is unlikely. Similarly, the cleavage and religation rates of the MCs were not topologically dependent, but topoisomers with low superhelical densities were found to unwind more slowly than highly supercoiled topoisomers, suggesting that reduced torque at low superhelical densities leads to an increased number of cycles of cleavage and ligation before a successful unwinding event. The K271E charge reversal mutant has an impaired interaction with the rotating DNA segment that leads to an increase in the number of supercoils that were unwound per cleavage event. This result provides evidence that interactions of the enzyme with the rotating DNA segment can restrict the number of supercoils that are unwound. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to be important in regulating the steady-state superhelical density of DNA domains in the cell.

Preventing the return of smallpox: molecular modeling studies on thymidylate kinase from Variola virus.

Smallpox was one of the most devastating diseases in the human history and still represents a serious menace today due to its potential use by bioterrorists. Considering this threat and the non-existence of effective chemotherapy, we propose the enzyme thymidylate kinase from Variola virus (VarTMPK) as a potential target to the drug design against smallpox. We first built a homology model for VarTMPK and performed molecular docking studies on it in order to investigate the interactions with inhibitors of Vaccinia virus TMPK (VacTMPK). Subsequently, molecular dynamics (MD) simulations of these compounds inside VarTMPK and human TMPK (HssTMPK) were carried out in order to select the most promising and selective compounds as leads for the design of potential VarTMPK inhibitors. Results of the docking and MD simulations corroborated to each other, suggesting selectivity towards VarTMPK and, also, a good correlation with the experimental data.

Activity, specificity, and probe design for the smallpox virus protease K7L.

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

Variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and SRC family tyrosine kinases.

Vaccinia virus (VacV) enters mammalian cells, replicates extranuclearly, and produces virions that move to the cell surface along microtubules, fuse with the plasma membrane, and move from infected cells toward apposing cells on actin-filled membranous protrusions or actin tails. To form actin tails, cell-associated enveloped virions (CEV) require Abl and Src family tyrosine kinases. Furthermore, release of CEV from the cell requires Abl but not Src family tyrosine kinases and is blocked by imatinib mesylate (STI-571; Gleevec), an Abl family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Here we demonstrate that the Poxviridae family members monkeypox virus (MPX) and variola virus (VarV) use conserved mechanisms for actin motility and extracellular enveloped virion (EEV) release. Furthermore, we show that imatinib mesylate is effective in a mouse model of infection with VacV, whether delivered prophylactically or postinfection, and restricts spread of virions from the site of inoculation. While inhibitors of both Src and Abl family kinases, such as dasatinib (BMS-354825; Sprycel), are effective in limiting dissemination of VacV, VarV, and MPX in vitro, members of this class of drugs appear to have immunosuppressive effects in vivo that preclude their use as anti-infectives. Together, these data suggest a possible utility for imatinib mesylate in treating smallpox or MPX infections or complications associated with vaccination.

Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic.

Poxviruses encode their own type IB topoisomerases (TopIBs), which release superhelical tension generated by replication and transcription of their genomes. To investigate the reaction catalyzed by viral TopIBs, we have determined the structure of a variola virus topoisomerase-DNA complex trapped as a vanadate transition state mimic. The structure reveals how the viral TopIB enzymes are likely to position the DNA duplex for ligation following relaxation of supercoils and identifies the sources of friction observed in single-molecule experiments that argue against free rotation. The structure also identifies a conformational change in the leaving group sugar that must occur prior to cleavage and reveals a mechanism for promoting ligation following relaxation of supercoils that involves an Asp-minor groove interaction. Overall, the new structural data support a common catalytic mechanism for the TopIB superfamily but indicate distinct methods for controlling duplex rotation in the small versus large enzyme subfamilies.

Structure-assisted discovery of Variola major H1 phosphatase inhibitors.

Variola major virus, the causative agent of smallpox, encodes the dual-specificity H1 phosphatase. Because this enzyme is essential for the production of mature virus particles, it is an attractive molecular target for the development of therapeutic countermeasures for this potential agent of bioterrorism. As a first step in this direction, the crystal structure of H1 phosphatase has been determined at a resolution of 1.8 A. In silico screening methods have led to the identification of several small molecules that inhibit Variola H1 phosphatase with IC(50) values in the low micromolar range. These molecules provide novel leads for future drug development.

Variola virus topoisomerase: DNA cleavage specificity and distribution of sites in Poxvirus genomes.

Topoisomerase enzymes regulate superhelical tension in DNA resulting from transcription, replication, repair, and other molecular transactions. Poxviruses encode an unusual type IB topoisomerase that acts only at conserved DNA sequences containing the core pentanucleotide 5'-(T/C)CCTT-3'. In X-ray structures of the variola virus topoisomerase bound to DNA, protein-DNA contacts were found to extend beyond the core pentanucleotide, indicating that the full recognition site has not yet been fully defined in functional studies. Here we report quantitation of DNA cleavage rates for an optimized 13 bp site and for all possible single base substitutions (40 total sites), with the goals of understanding the molecular mechanism of recognition and mapping topoisomerase sites in poxvirus genome sequences. The data allow a precise definition of enzyme-DNA interactions and the energetic contributions of each. We then used the resulting "action matrix" to show that favorable topoisomerase sites are distributed all along the length of poxvirus DNA sequences, consistent with a requirement for local release of superhelical tension in constrained topological domains. In orthopox genomes, an additional central cluster of sites was also evident. A negative correlation of predicted topoisomerase sites was seen relative to early terminators, but no correlation was seen with early or late promoters. These data define the full variola virus topoisomerase recognition site and provide a new window on topoisomerase function in vivo.

Regulation of catalysis by the smallpox virus topoisomerase.

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp(168) is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.

Unraveling the structure of the variola topoisomerase IB-DNA complex: a possible new twist on smallpox therapy.

Smallpox is a serious and highly contagious disease that is caused by the variola virus. It is one of the most severe infectious human diseases known, with mortality rates as high as 30%. A successful worldwide vaccination program led to the eradication of smallpox in 1980. However, the high transmission rate of variola virus, coupled with the deadly nature of smallpox, makes this virus a potentially devastating weapon for bioterrorism. Currently, there is no specific treatment for smallpox. However, a recent article on the structure of a variola topoisomerase IB-DNA complex provides an intriguing starting point for the rational design of drugs with potential activity against smallpox.

Structural basis for specificity in the poxvirus topoisomerase.

Although smallpox has been eradicated from the human population, it is presently feared as a possible agent of bioterrorism. The smallpox virus codes for its own topoisomerase enzyme that differs from its cellular counterpart by requiring a specific DNA sequence for activation of catalysis. Here we present crystal structures of the smallpox virus topoisomerase enzyme bound both covalently and noncovalently to a specific DNA sequence. These structures reveal the basis for site-specific DNA recognition, and they explain how catalysis is likely activated by formation of a specific enzyme-DNA interface. Unexpectedly, the poxvirus enzyme uses a major groove binding alpha helix that is not present in the human enzyme to recognize part of the core recognition sequence and activate the enzyme for catalysis. The topoisomerase-DNA complex structures also provide a three-dimensional framework that may facilitate the rational design of therapeutic agents to treat poxvirus infections.

Looking back at smallpox.

Smallpox apparently arose through transfer of variola virus to humans from another animal species. By causing a brief infection that required close contact for transmission and engendered solid immunity, the agent was always vulnerable to simple isolation measures. The high replicative fidelity of the viral DNA polymerase limited variola's ability to adapt to humans and preserved orthopoxviral antigenic cross-reactivity, so that vaccinia vaccination protected against smallpox. Host-derived genes encoding immunomodulatory proteins helped shelter viral replication from innate immune responses. Examination of clinical variants suggests that severity of illness was usually determined by host responses during the incubation period. Control of viral replication was aided by early postexposure vaccination and might be strengthened by additional immunological interventions. Massive inflammatory responses were responsible for major features of illness. Some patients with high levels of circulating virus developed hemorrhagic disease resembling septic shock. Continued study of virus-host interactions is needed to defend against genetically modified agents.

Two types of deletions in orthopoxvirus genomes.

The genome nucleotide sequences of two strains of variola major virus and one strain of vaccinia virus were compared. One hundred and sixty-eight short (less than 100 bp in length) and eight long (more than 900 bp in length) deletions, four deletion/insertion regions, and four regions of multiple mutational differences between variola and vaccinia virus DNAs were revealed. Short deletions generally occur at directly repeated sequences of 3-21 bp. Long deletions showed no evidence of repeated sequences at their points of junction. We suggest the presence of a consensus sequence characteristic of these junctions and propose that there is a virus-encoded enzyme that produces this nonhomologous recombination/deletion in the cytoplasm of the infected cell.

Analysis of the nucleotide sequence of 53 kbp from the right terminus of the genome of variola major virus strain India-1967.

Sequencing and computer analysis of a variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region has been carried out. Fifty-nine potential open reading frames (ORFs) of over 60 amino acid residues were identified. Structure-function organization of the VAR-IND DNA segment was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple differences between VAR-IND and the strains of VAC but the high identity of VAR-IND with VAR-HAR in the genetic maps are revealed. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.

Nucleotide sequence of the thymidine kinase gene region of monkeypox and variola viruses.

Among the orthopoxviruses variola virus induces in cells a characteristic thymidine kinase (TK) activity that can be feedback inhibited in reactions with thymidine triphosphate. Northern blot analyses of variola and monkeypox virus-infected cell extracts showed RNAs of the same molecular weight as the major (590-base) and minor (2380-base) TK transcripts described for vaccinia virus. The nucleotide sequences of 1275 bp in the TK gene region of variola and monkeypox viruses have been determined. When these sequences were compared with such sequences reported for vaccinia virus, differences were observed at 41 nucleotide positions. Examination of the putative encoded TK polypeptide for the three viruses revealed variation at eight amino acid positions. Two major differences in the amino acid composition of the variola virus TK were identified that might play a role in alteration of its kinetic properties.