ABSTRACT
Transposases are ubiquitous mobile genetic elements responsible for genome development, driving rearrangements, such as insertions, deletions and translocations. Across species evolution, some transposases are tamed by their host and are made part of complex cellular systems. The proliferation of retroviruses is also dependent on transposase related enzymes termed integrases. Recombinationactivating gene protein (RAG)1 and metnase are just two examples of transposase domestication and together with retroviral integrases (INs), they belong to the DDE polynucleotidyl transferases superfamily. They share mechanistic and structural features linked to the RNase Hlike fold, harboring a DDE(D) metal dependent catalytic motif. Recent antiretroviral compounds target the catalytic domain of integrase, but they also have the potential of inhibiting other related enzymes. In this review, we report the activity of different classes of integrase inhibitors on various DDE transposases. Computational simulations are useful to predict the extent of offtarget activity and have been employed to study the interactions between RAG1 recombinase and compounds from three different pharmacologic classes. We demonstrate that strandtransfer inhibitors display a higher affinity towards the RAG1 RNase H domain, as suggested by experimental data compared to allosteric inhibitors. While interference with RAG1 and 2 recombination is associated with a negative impact on immune function, the inhibition of metnase or HTLV1 integrase opens the way for the development of novel therapies for refractory cancers.
Subject(s)
DNA-Binding Proteins/metabolism , Dichlorodiphenyl Dichloroethylene , HIV-1/genetics , Homeodomain Proteins/metabolism , Integrase Inhibitors/pharmacology , Nuclear Proteins/metabolism , Recombination, Genetic/genetics , Transposases/drug effects , Catalytic Domain , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , HIV-1/metabolism , Heterocyclic Compounds, 3-Ring , Histone-Lysine N-Methyltransferase , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Integrase Inhibitors/chemistry , Molecular Docking Simulation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Oxazines , Piperazines , Protein Conformation , Pyridones , Retroviridae/genetics , Ribonuclease H, Human Immunodeficiency Virus/metabolismABSTRACT
Diketoacid (DKA) compounds have been shown to inhibit HIV-1 integrase by a mechanism that involves sequestration of the active site metals. Because HIV-1 integrase and Tn5 transposase have similar active site architectures and catalytic mechanisms, we investigated whether DKA analogues would inhibit Tn5 transposase activity and provide a model system to explore the mechanisms of action of these inhibitors. A screen of several hundred DKA analogues identified several with activity against Tn5 Tnp. Six DKA inhibitors used in this study manifested a variety of effects on different transposition steps suggesting that different analogues may have different binding contacts with transposase. All DKA compounds inhibited paired end complex (PEC) formation in which the nucleoprotein complex required for catalysis is assembled. Dissociation of PECs by some DKA compounds indicates that these inhibitors can decrease PEC stability. Four DKA compounds inhibited the two cleavage steps releasing transposon DNA from flanking DNA, and one of these four compounds preferentially inhibited the second cleavage step. The differential effect of this inhibitor on the second cleavage event indicates that cleavage of the two transposon-donor DNA boundaries is a sequential process requiring a conformational change. The requirement for a conformational change between cleavage events was also demonstrated by the inability of transposase to perform second cleavage at 25 degrees C. Finally, all six compounds inhibit strand transfer, the final step of Tn5 transposition. Two of the compounds that inhibited strand transfer have no effect on DNA cleavage. The strand transfer inhibition properties of various DKA compounds was sensitive to the structure of the 5'-non-transferred strand, suggesting that these compounds bind in or near the transposase active site. Other results that probe compound binding sites include the effects of active site mutations and donor DNA on DKA compound inhibition activities. Thus, DKA inhibitors will provide an important set of tools to investigate the mechanism of action of transposases and integrases.
Subject(s)
DNA-Binding Proteins/genetics , HIV Integrase Inhibitors/pharmacology , HIV-1/enzymology , Keto Acids/chemistry , Models, Molecular , Transposases/drug effects , Transposases/genetics , Anti-HIV Agents/chemistry , Base Sequence , Binding Sites , Catalysis/drug effects , DNA Transposable Elements/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , HIV Integrase/chemistry , HIV Integrase/drug effects , HIV Integrase/metabolism , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/metabolism , HIV-1/genetics , Keto Acids/metabolism , Keto Acids/pharmacology , Magnesium/chemistry , Magnesium/metabolism , Molecular Structure , Nucleic Acid Conformation , Oligonucleotides/antagonists & inhibitors , Oligonucleotides/genetics , Oligonucleotides/metabolism , Point Mutation , Protein Binding , Protein Conformation , Transposases/metabolismABSTRACT
Human immunodeficiency virus (HIV) type 1 (HIV-1) integrase is an underutilized drug target for the treatment of HIV infection. One limiting factor is the lack of costructural data for use in the rational design or modification of integrase inhibitors. Tn5 transposase is a structurally well characterized, related protein that may serve as a useful surrogate. However, little data exist on inhibitor cross-reactivity. Here we screened 16,000 compounds using Tn5 transposase as the target and identified 20 compounds that appear to specifically inhibit complex assembly. Six were found to also inhibit HIV-1 integrase. These compounds likely interact with a highly conserved region presumably within the catalytic core. Most promising, several cinnamoyl derivatives were found to inhibit HIV transduction in cells. The identification of integrase inhibitors from a screen using Tn5 transposase as the target illustrates the utility of Tn5 as a surrogate for HIV-1 integration even though the relationship between the two systems is limited to the active site architecture and catalytic mechanism.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , Transposases/drug effects , Cell Survival/drug effects , Cross Reactions , DNA, Viral/biosynthesis , DNA, Viral/genetics , Drug Design , Drug Evaluation, Preclinical , Electrophoretic Mobility Shift Assay , HIV Integrase Inhibitors/pharmacology , HIV-1/growth & development , Humans , Models, Molecular , Peptide Library , Structure-Activity Relationship , Transduction, GeneticABSTRACT
The dif locus is a site-specific recombination site located within the terminus region of the chromosome of Escherichia coli. Recombination at dif resolves circular dimer chromosomes to monomers, and this recombination requires the XerC, XerD and FtsK proteins, as well as cell division. In order to characterize other enzymes that interact at dif, we tested whether quinolone-induced cleavage occurs at this site. Quinolone drugs, such as norfloxacin, inhibit the type 2 topoisomerases, DNA gyrase and topoisomerase IV, and can cleave DNA at sites where these enzymes interact with the chromosome. Using strains in which either DNA gyrase or topoisomerase IV, or both, were resistant to norfloxacin, we determined that specific interactions between dif and topoisomerase IV caused cleavage at that site. This interaction required XerC and XerD, but did not require the C-terminal region of FtsK or cell division.
