RESUMO
Biological applications of phosphorescent probes for sensing molecular oxygen (O2) and bioimaging have gained popularity, but their choice is rather limited. We describe a family of new heterosubstituted phosphorescent bioprobes based on the Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye. The probes are produced by simple click modification of its para-fluorine atoms with thiols, such as 1/2-thio-glucose, thio-poly(ethylene glycol) (PEG), or cysteamine. The probes were designed to have one cell-targeting moiety and three polar moieties forming a hydrophilic shell. Their chemical synthesis and purification were optimized to produce high reaction yields and easy scale-up. The ability to perform as cell-permeable or -impermeable probes was tuned by the polarity and molecular charge of the bioconjugate. The new PtPFPP derivatives were characterized for their spectral properties and cell-penetrating ability in the experiments with mammalian cell cultures, using a time-resolved fluorescence reader and PLIM imaging detection. Structure-activity relationships were established. Thus, the tri- and tetra-PEGylated structures showed low cell internalization allowing their use as extracellular probes, while cysteamine derivatives performed as efficient intracellular probes. No significant cytotoxicity was observed for all of the probes under the experimental conditions used.
Assuntos
Técnicas Biossensoriais , Porfirinas , Animais , Cisteamina , Porfirinas/química , Oxigênio , Técnicas Biossensoriais/métodos , Relação Estrutura-Atividade , MamíferosRESUMO
The synthesis of guanine α-carboxy nucleoside phosphonate (G-α-CNP) is described. Two routes provide access to racemic G-α-CNP 9, one via base construction and the other utilizing Tsuji-Trost allylic substitution. The latter methodology was also applied to the enantiopure synthesis of both antipodes of G-α-CNP, each of which showing interesting antiviral DNA polymerase activity. Additionally, we report an improved multigram scale preparation of the cyclopentene building block 10, starting material for the preferred Tsuji-Trost route to 9.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Guanina/química , Inibidores da Síntese de Ácido Nucleico/síntese química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Organofosfonatos/síntese química , Organofosfonatos/farmacologia , Nucleosídeos de Purina/química , Catálise , Técnicas de Química Sintética , HIV-1/enzimologia , Inibidores da Síntese de Ácido Nucleico/química , Organofosfonatos/química , Paládio/químicaRESUMO
Polymerases have a structurally highly conserved negatively charged amino acid motif that is strictly required for Mg(2+) cation-dependent catalytic incorporation of (d)NTP nucleotides into nucleic acids. Based on these characteristics, a nucleoside monophosphonate scaffold, α-carboxy nucleoside phosphonate (α-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that α-CNPs mimic the dNTP binding through a carboxylate oxygen, two phosphonate oxygens, and base-pairing with the template. In particular, the carboxyl oxygen of the α-CNP acts as the potential equivalent of the α-phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the α-CNP chelate Mg(2+), mimicking the chelation by the ß- and γ-phosphate oxygens of dNTPs. α-CNPs (i) do not require metabolic activation (phosphorylation), (ii) bind directly to the substrate-binding site, (iii) chelate one of the two active site Mg(2+) ions, and (iv) reversibly inhibit the polymerase catalytic activity without being incorporated into nucleic acids. In addition, α-CNPs were also found to selectively interact with regulatory (i.e., allosteric) Mg(2+)-dNTP-binding sites of nucleos(t)ide-metabolizing enzymes susceptible to metabolic regulation. α-CNPs represent an entirely novel and broad technological platform for the development of specific substrate active- or regulatory-site inhibitors with therapeutic potential.
Assuntos
Nucleosídeos/farmacologia , Nucleotídeos/farmacologia , Organofosfonatos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sequência de Bases , Biocatálise/efeitos dos fármacos , Extratos Celulares , DNA Polimerase Dirigida por DNA/metabolismo , Farmacorresistência Viral/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Células HeLa , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Nucleosídeos/química , Nucleotídeos/química , Organofosfonatos/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , EstereoisomerismoRESUMO
As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 reverse transcriptase inhibition, structurally related derivatives were synthesized, namely the malonate 2, the unsaturated and saturated bisphosphonates 3 and 4, respectively and the amide 5. These compounds were evaluated for inhibition of HIV-1 reverse transcriptase in cell-free assays. The importance of the α-carboxy phosphonoacetic acid moiety for achieving reverse transcriptase inhibition, without the need for prior phosphorylation, was confirmed. The malonate derivative 2 was less active by two orders of magnitude than the original α-CNPs, while displaying the same pattern of kinetic behavior; interestingly the activity resides in the "L"-enantiomer of 2, as seen with the earlier series of α-CNPs. A crystal structure with an RT/DNA complex at 2.95 Å resolution revealed the binding of the "L"-enantiomer of 2, at the polymerase active site with a weaker metal ion chelation environment compared to 1a (T-α-CNP) which may explain the lower inhibitory activity of 2.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Nucleosídeos/farmacologia , Organofosfonatos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/síntese química , Relação Dose-Resposta a Droga , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Modelos Moleculares , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Organofosfonatos/síntese química , Organofosfonatos/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Relação Estrutura-AtividadeRESUMO
The synthesis of the first series of a new class of nucleoside phosphonate analogues is described. Addition of a carboxyl group at the α position of carbocyclic nucleoside phosphonate analogues leads to a novel class of potent HIV reverse transcriptase (RT) inhibitors, α-carboxy nucleoside phosphonates (α-CNPs). Key steps in the synthesis of the compounds are Rh-catalyzed O-H insertion and Pd-catalyzed allylation reactions. In cell-free assays, the final products are markedly inhibitory against HIV RT and do not require phosphorylation to exhibit anti-RT activity, which indicates that the α-carboxyphosphonate function is efficiently recognized by HIV RT as a triphosphate entity, an unprecedented property of nucleoside monophosph(on)ates.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , HIV-1/efeitos dos fármacos , Nucleosídeos/síntese química , Organofosfonatos/síntese química , Organofosfonatos/farmacologia , Paládio/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/síntese química , Antivirais/química , Catálise , Desenho de Fármacos , Humanos , Modelos Moleculares , Nucleosídeos/farmacologia , Organofosfonatos/química , Fosforilação , Relação Estrutura-AtividadeRESUMO
Acyclic nucleoside phosphonates represent a well-defined class of clinically used nucleoside analogs. All acyclic nucleoside phosphonates need intracellular phosphorylation before they can bind viral DNA polymerases. Recently, a novel class of alpha-carboxynucleoside phosphonates have been designed to mimic the natural 2'-deoxynucleotide 5'-triphosphate substrates of DNA polymerases. They contain a carboxyl group in the phosphonate moiety linked to the nucleobase through a cyclic or acyclic bridge. Alpha-carboxynucleoside phosphonates act as viral DNA polymerase inhibitors without any prior requirement of metabolic conversion. Selective inhibitory activity against retroviral reverse transcriptase and herpesvirus DNA polymerases have been demonstrated. These compounds have a unique mechanism of inhibition of viral DNA polymerases, and provide possibilities for further modifications to optimize and fine tune their antiviral DNA polymerase spectrum.
Assuntos
Antivirais/química , Antivirais/farmacologia , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleosídeos/análogos & derivados , Nucleosídeos/farmacologia , Organofosfonatos/química , Organofosfonatos/farmacologia , Animais , DNA Polimerase Dirigida por DNA , Descoberta de Drogas , Exodesoxirribonucleases/antagonistas & inibidores , Herpes Simples/tratamento farmacológico , Humanos , Modelos Moleculares , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Proteínas Virais/antagonistas & inibidores , Viroses/tratamento farmacológico , Vírus/efeitos dos fármacos , Vírus/enzimologiaRESUMO
α-Carboxy nucleoside phosphonates (α-CNPs) are modified nucleotides that represent a novel class of nucleotide-competing reverse transcriptase (RT) inhibitors (NcRTIs). They were designed to act directly against HIV-1 RT without the need for prior activation (phosphorylation). In this respect, they differ from the nucleoside or nucleotide RTIs [N(t)RTIs] that require conversion to their triphosphate forms before being inhibitory to HIV-1 RT. The guanine derivative (G-α-CNP) has now been synthesized and investigated for the first time. The (L)-(+)-enantiomer of G-α-CNP directly and competitively inhibits HIV-1 RT by interacting with the substrate active site of the enzyme. The (D)-(-)-enantiomer proved inactive against HIV-1 RT. In contrast, the (+)- and (-)-enantiomers of G-α-CNP inhibited herpes (i.e. HSV-1, HCMV) DNA polymerases in a non- or uncompetitive manner, strongly indicating interaction of the (L)-(+)- and the (D)-(-)-G-α-CNPs at a location different from the polymerase substrate active site of the herpes enzymes. Such entirely different inhibition profile of viral polymerases is unprecedented for a single antiviral drug molecule. Moreover, within the class of α-CNPs, subtle differences in their sensitivity to mutant HIV-1 RT enzymes were observed depending on the nature of the nucleobase in the α-CNP molecules. The unique properties of the α-CNPs make this class of compounds, including G-α-CNP, direct acting inhibitors of multiple viral DNA polymerases.