HIV-1 Integrase-Targeted Short Peptides Derived from a Viral Protein R Sequence.

HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69⁻75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site.

6,7-Dihydroxyisoindolin-1-one and 7,8-Dihydroxy-3,4-Dihydroisoquinolin- 1(2H)-one Based HIV-1 Integrase Inhibitors.

Integrase (IN) is an essential viral enzyme required for HIV-1 replication, which has been targeted by anti-AIDS therapeutics. Integrase strand transfer inhibitors (INSTIs) represent a new class of antiretroviral agents developed for the treatment of HIV-1 infections. Important structural features that are shared by many INSTIs include a coplanar arrangement of three heteroatoms that chelate two catalytic Mg(2+) ions in the IN active site and a linked halophenyl ring that binds in the hydrophobic pocket formed by the complex of IN with viral DNA. We recently reported bicyclic 6,7-dihydroxyoxoisoindolin-1-one-based IN inhibitors. In the current study, we modified these inhibitors in three ways. First, we increased the spacer length between the metalchelating triad and the halophenyl group. Second, we replaced the indoline [5,6] bicycle with a fused dihydroxyisoquinolinones [6,6] ring system. Finally, we prepared bis-6,7-dihydroxyisoindolin-1-one-4-sulfonamides as dimeric HIV-1 IN inhibitors. These new analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays.

Basic quinolinonyl diketo acid derivatives as inhibitors of HIV integrase and their activity against RNase H function of reverse transcriptase.

A series of antiviral basic quinolinonyl diketo acid derivatives were developed as inhibitors of HIV-1 IN. Compounds 12d,f,i inhibited HIV-1 IN with IC50 values below 100 nM for strand transfer and showed a 2 order of magnitude selectivity over 3'-processing. These strand transfer selective inhibitors also inhibited HIV-1 RNase H with low micromolar potencies. Molecular modeling studies based on both the HIV-1 IN and RNase H catalytic core domains provided new structural insights for the future development of these compounds as dual HIV-1 IN and RNase H inhibitors.

Cell-permeable stapled peptides based on HIV-1 integrase inhibitors derived from HIV-1 gene products.

HIV-1 integrase (IN) is an enzyme which is indispensable for the stable infection of host cells because it catalyzes the insertion of viral DNA into the genome and thus is an attractive target for the development of anti-HIV agents. Earlier, we found Vpr-derived peptides with inhibitory activity against HIV-1 IN. These Vpr-derived peptides are originally located in an α-helical region of the parent Vpr protein. Addition of an octa-arginyl group to the inhibitory peptides caused significant inhibition against HIV replication associated with an increase in cell permeability but also relatively high cytotoxicity. In the current study, stapled peptides, a new class of stabilized α-helical peptidomimetics were adopted to enhance the cell permeability of the above lead peptides. A series of stapled peptides, which have a hydrocarbon link formed by a ruthenium-catalyzed ring-closing metathesis reaction between successive turns of α-helix, were designed, synthesized, and evaluated for biological activity. In cell-based assays some of the stapled peptides showed potent anti-HIV activity comparable with that of the original octa-arginine-containing peptide (2) but with lower cytotoxicity. Fluorescent imaging experiments revealed that these stapled peptides are significantly cell permeable, and CD analysis showed they form α-helical structures, whereas the unstapled congeners form ß-sheet structures. The application of this stapling strategy to Vpr-derived IN inhibitory peptides led to a remarkable increase in their potency in cells and a significant reduction of their cytotoxicity.

Activities, crystal structures, and molecular dynamics of dihydro-1H-isoindole derivatives, inhibitors of HIV-1 integrase.

On the basis of a series of lactam and phthalimide derivatives that inhibit HIV-1 integrase, we developed a new molecule, XZ-259, with biochemical and antiviral activities comparable to raltegravir. We determined the crystal structures of XZ-259 and four other derivatives in complex with the prototype foamy virus intasome. The compounds bind at the integrase-Mg(2+)-DNA interface of the integrase active site. In biochemical and antiviral assays, XZ-259 inhibits raltegravir-resistant HIV-1 integrases harboring the Y143R mutation. Molecular modeling is also presented suggesting that XZ-259 can bind in the HIV-1 intasome with its dimethyl sulfonamide group adopting two opposite orientations. Molecular dynamics analyses of the HIV-1 intasome highlight the importance of the viral DNA in drug potency.

6,7-Dihydroxy-1-oxoisoindoline-4-sulfonamide-containing HIV-1 integrase inhibitors.

Although an extensive body of scientific and patent literature exists describing the development of HIV-1 integrase (IN) inhibitors, Merck's raltegravir and Gilead's elvitegravir remain the only IN inhibitors FDA-approved for the treatment of AIDS. The emergence of raltegravir-resistant strains of HIV-1 containing mutated forms of IN underlies the need for continued efforts to enhance the efficacy of IN inhibitors against resistant mutants. We have previously described bicyclic 6,7-dihydroxyoxoisoindolin-1-ones that show good IN inhibitory potency. This report describes the effects of introducing substituents into the 4- and 5-positions of the parent 6,7-dihydroxyoxoisoindolin-1-one platform. We have developed several sulfonamide-containing analogs that enhance potency in cell-based HIV assays by more than two orders-of-magnitude and we describe several compounds that are more potent than raltegravir against the clinically relevant Y143R IN mutant.

Probing chelation motifs in HIV integrase inhibitors.

A series of HIV integrase (HIV-1 IN) inhibitors were synthesized to evaluate the role of the metal-binding group (MBG) in this class of metalloenzyme inhibitors. A total of 21 different raltegravir-chelator derivative (RCD) compounds were prepared that differed only in the nature of the MBG. These IN strand-transfer inhibitors (INSTIs) were evaluated in vitro in cell-free enzyme activity assays, and the in vitro results were further validated in cell culture experiments. All of the active compounds showed selective inhibition of the strand-transfer reaction over 3'-processing, suggesting a common mode of action with raltegravir. The results of the in vitro activity suggest that the nature of the MBG donor atoms, the overall MBG structure, and the specific arrangement of the MBG donor atom triad are essential for obtaining maximal HIV-1 IN inhibition. At least two compounds (RCD-4, RCD-5) containing a hydroxypyrone MBG were found to display superior strand-transfer inhibition when compared to an abbreviated analogue of raltegravir (RCD-1). By isolating and examining the role of the MBG in a series of INSTIs, we have identified a scaffold (hydroxypyrones) that may provide access to a unique class of HIV-1 IN inhibitors, and may help overcome rising raltegravir resistance.

Bicyclic hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhibitors.

HIV-1 integrase (IN) is a validated therapeutic target for the treatment of AIDS. However, the emergence of resistance to raltegravir, the sole marketed FDA-approved IN inhibitor, emphasizes the need to develop second-generation inhibitors that retain efficacy against clinically relevant IN mutants. We report herein bicyclic hydroxy-1H-pyrrolopyridine-triones as a new family of HIV-1 integrase inhibitors that were efficiently prepared using a key 'Pummerer cyclization deprotonation cycloaddition' cascade of imidosulfoxides. In in vitro HIV-1 integrase assays, the analogs showed low micromolar inhibitory potencies with selectivity for strand transfer reactions as compared with 3'-processing inhibition. A representative inhibitor (5e) retained most of its inhibitory potency against the three major raltegravir-resistant IN mutant enzymes, G140S/Q148H, Y143R, and N155H. In antiviral assays employing viral vectors coding these IN mutants, compound 5e was approximately 200- and 20-fold less affected than raltegravir against the G140S/Q148H and Y143R mutations, respectively. Against the N155H mutation, 5e was approximately 10-fold less affected than raltegravir. Thus, our new compounds represent a novel structural class that may be further developed to overcome resistance to raltegravir, particularly in the case of the G140S/Q148H mutations.

Development of tricyclic hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhibitors.

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3'-processing.

N-3 Hydroxylation of Pyrimidine-2,4-diones Yields Dual Inhibitors of HIV Reverse Transcriptase and Integrase.

A new molecular scaffold featuring an N-hydroxyimide functionality and capable of inhibiting both reverse transcriptase (RT) and integrase (IN) of Human Immunodeficiency Virus (HIV) was rationally designed based on 1-[(2-hydroxyethoxy) methyl]-6-(phenylthio)-thymine (HEPT) non-nucleoside RT inhibitors (NNRTIs). The design involves a minimal 3-N hydroxylation of the pyrimidine ring of HEPT compound to yield a chelating triad which, along with the existing benzyl group, appeared to satisfy major structural requirements for IN binding. In the mean time, this chemical modification did not severely compromise the compound's ability to inhibit RT. A preliminary structure-activity-relationship (SAR) study reveals that this N-3 OH is essential for IN inhibition and that the benzyl group on N-1 side chain is more important for IN binding than the one on C-6.

Elvitegravir overcomes resistance to raltegravir induced by integrase mutation Y143.

OBJECTIVE: In this study, we characterized elvitegravir activity in the context of raltegravir resistance mutations. DESIGN: Using site-directed mutagenesis, we generated recombinant integrase proteins and viruses harboring raltegravir resistance mutation to assess the biochemical and cellular activity of elvitegravir in the presence of such mutants. METHODS: Recombinant proteins were used in gel-based assays. Antiviral data were obtained with reporter viruses in a single-round infection using a luciferase-based assay. RESULTS: Although main raltegravir resistance pathways involving mutations at integrase position 148 and 155 confer cross-resistance to elvitegravir, elvitegravir remains fully active against the Y143R mutant integrase and virus particles. CONCLUSION: In addition to favorable pharmacokinetics compared to raltegravir, our findings provide the rationale for using elvitegravir in patients failing raltegravir because of the integrase mutation Y143.

3-Hydroxypyrimidine-2,4-diones as an inhibitor scaffold of HIV integrase.

Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). Inhibitors with a novel structure core are essential for combating resistance associated with known IN inhibitors (INIs). We have previously disclosed a novel dual inhibitor scaffold of HIV IN and reverse transcriptase (RT). Here we report the complete structure-activity relationship (SAR), molecular modeling, and resistance profile of this inhibitor type on IN inhibition. These studies support an antiviral mechanism of dual inhibition against both IN and RT and validate 3-hydroxypyrimidine-2,4-diones as an IN inhibitor scaffold.

6-Benzoyl-3-hydroxypyrimidine-2,4-diones as dual inhibitors of HIV reverse transcriptase and integrase.

N-3-hydroxylation of pyrimidine-2,4-diones was recently found to yield inhibitors of both HIV-1 reverse transcriptase (RT) and integrase (IN). An extended series of analogues featuring a benzoyl group at the C-6 position of the pyrimidine ring was synthesized. Through biochemical studies it was found that these new analogues are dually active against both RT and IN in low micromolar range. Antiviral assays confirmed that these new inhibitors are active against HIV-1 in cell culture at nanomolar to low micromolar range, further validating 3-hydroxypyrimidine-2,4-diones as a viable scaffold for antiviral development.

Role of tyrosyl-DNA phosphodiesterase (TDP1) in mitochondria.

Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3'-end linked to a tyrosyl moiety and has been implicated in the repair of topoisomerase I (Top1)-DNA covalent complexes. TDP1 can also hydrolyze other 3'-end DNA alterations including 3'-phosphoglycolate and 3'-abasic sites, and exhibits 3'-nucleosidase activity indicating it may function as a general 3'-end-processing DNA repair enzyme. Here, using laser confocal microscopy, subcellular fractionation and biochemical analyses we demonstrate that a fraction of the TDP1 encoded by the nuclear TDP1 gene localizes to mitochondria. We also show that mitochondrial base excision repair depends on TDP1 activity and provide evidence that TDP1 is required for efficient repair of oxidative damage in mitochondrial DNA. Together, our findings provide evidence for TDP1 as a novel mitochondrial enzyme.

Preparation and characterization of novel 4-bromo-3,4-dimethyl-1-phenyl-2-phospholene 1-oxide and the analogous phosphorus heterocycles or phospha sugars.

4-Bromo-3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (3c) was first synthesized from 3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (2c) by a bromo-radical substitution reaction occurred at C-4 position by N-bromosuccinimide and 2,2'-azobisisobutyronitrile. The novel phospha sugar analogue 3c exerted high anti-proliferative effect on U937 cells evaluated by MTT in vitro methods and was much more efficient than that of Gleevec, which is known as a molecule targeting chemotherapeutical agent. The substitution of 2-phospholenes at C-3 and C-4 position with methyl groups as well as 4-bromo substituent suggests a good anti-proliferative effect.

Peptidic HIV integrase inhibitors derived from HIV gene products: structure-activity relationship studies.

Structure-activity relationship studies were conducted on HIV integrase (IN) inhibitory peptides which were found by the screening of an overlapping peptide library derived from HIV-1 gene products. Since these peptides located in the second helix of Vpr are considered to have an alpha-helical conformation, Glu-Lys pairs were introduced into the i and i+4 positions to increase the helicity of the lead compound possessing an octa-arginyl group. Ala-scan was also performed on the lead compound for the identification of the amino acid residues responsible for the inhibitory activity. The results indicated the importance of an alpha-helical structure for the expression of inhibitory activity, and presented a binding model of integrase and the lead compound.

Resistance to integrase inhibitors.

Integrase (IN) is a clinically validated target for the treatment of human immunodeficiency virus infections and raltegravir exhibits remarkable clinical activity. The next most advanced IN inhibitor is elvitegravir. However, mutant viruses lead to treatment failure and mutations within the IN coding sequence appear to confer cross-resistance. The characterization of those mutations is critical for the development of second generation IN inhibitors to overcome resistance. This review focuses on IN resistance based on structural and biochemical data, and on the role of the IN flexible loop i.e., between residues G140-G149 in drug action and resistance.

Peptide HIV-1 integrase inhibitors from HIV-1 gene products.

Anti-HIV peptides with inhibitory activity against HIV-1 integrase (IN) have been found in overlapping peptide libraries derived from HIV-1 gene products. In a strand transfer assay using IN, inhibitory active peptides with certain sequential motifs related to Vpr- and Env-derived peptides were found. The addition of an octa-arginyl group to the inhibitory peptides caused a remarkable inhibition of the strand transfer and 3'-end-processing reactions catalyzed by IN and significant inhibition against HIV replication.

Scaffold rearrangement of dihydroxypyrimidine inhibitors of HIV integrase: Docking model revisited.

A series of dihydroxypyrimidine (DHP) derivatives were designed as inhibitors of HIV integrase (IN) based on known homology models. Through chemical synthesis and biochemical assays it was found that the activity profile of these compounds largely deviates from predictions with existing models. With the recently disclosed IN crystal structure of prototype foamy virus (PFV), a new HIV IN homology model was constructed featuring a critical IN/DNA interface previously lacking. With this new model, docking results completely corroborated observed biological activities. This new model should provide a more accurate and improved platform for the design of new inhibitors of HIV IN.

Biochemical and pharmacological analyses of HIV-1 integrase flexible loop mutants resistant to raltegravir.

Resistance to raltegravir (RAL), the first HIV-1 integrase (IN) inhibitor approved by the FDA, involves three genetic pathways: IN mutations N155H, Q148H/R/K, and Y143H/R/C. Those mutations are generally associated with secondary point mutations. The resulting mutant viruses show a high degree of resistance against RAL but somehow are affected in their replication capacity. Clinical and virological data indicate the high relevance of the combination G140S + Q148H because of its limited impact on HIV replication and very high resistance to RAL. Here, we report how mutations at the amino acid residues 140, 148, and 155 affect IN enzymatic activity and RAL resistance. We show that single mutations at position 140 have limited impact on 3'-processing (3'-P) but severely inactivate strand transfer (ST). On the other hand, single mutations at position 148 have a more profound effect and inactivate both 3'-P and ST. By examining systematically all of the double mutants at the 140 and 148 positions, we demonstrate that only the combination G140S + Q148H is able to restore the catalytic properties of IN. This rescue only operates in cis when both the 140S and 148H mutations are in the same IN polypeptide flexible loop. Finally, we show that the G140S-Q148H double mutant exhibits the highest resistance to RAL. It also confers cross-resistance to elvitegravir but less to G-quadraduplex inhibitors such as zintevir. Our results demonstrate that IN mutations at positions 140 and 148 in the IN flexible loop can account for the phenotype of RAL-resistant viruses.