ABSTRACT
Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Several different strategies for inactivation of RNRs have been reported, including the use of substrate analogues as mechanism-based inhibitors. This article undergoes a critical analysis on the current status of ribonucleotide reductase inhibitory mechanisms by substrate analogues highlighting experimental and theoretical/computational approaches. We have summarized a general portrait of the inhibitory mechanisms and classified the nucleoside analogue inhibitors in three main classes. The critical analysis undertaken will contribute in finding new and more effective ways of inhibiting RNR.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Nucleosides/chemistry , Nucleosides/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/chemistry , Humans , Models, BiologicalABSTRACT
HIV-1 protease is a crucial enzyme for the life cycle of the human immunodeficiency virus, the retrovirus that triggers AIDS. It is well documented that HIV-1 protease mediates the cleavage of Gag, Gag-Pol, and Nef precursor polyproteins and is highly selective concerning the set of 12 different amino acid sequences that cleaves. However, the governing principles and physical parameters, which determine substrate recognition and specificity, remain poorly understood despite the many speculative proposals that abound in the literature. In fact, it has been difficult so far to circumvent the fact that protease's substrates share little sequence identity and lack an obvious consensus binding motif. We have used microsecond time scale MD simulations to quantitatively show that some sequences of the polyprotein Gag-Pol that are not cleaved (nonsubstrates) have in fact a higher affinity to the active site of HIV-1 protease than a substrate; i.e., recognition is not governed by affinity to the active site. On the basis of a detailed analysis of the results and experimental data, we propose that the recognition is based on the geometric specificity of PR:Gag and PR:Gag-Pol multiprotein complex, that selects which residues lie in the specific position that makes them accessible to the active site for cleavage.
Subject(s)
HIV Protease/chemistry , Amino Acid Sequence , Catalytic Domain , Computational Biology , Humans , Molecular Dynamics Simulation , Molecular Sequence Data , Protein Structure, Tertiary , Substrate Specificity , Thermodynamics , gag Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/chemistry , pol Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. The enzyme has thus an attractive target for chemotherapies that fight proliferation-based diseases. 2'-Deoxy-2'methylenecytidine-5'-diphosphate (CH2dCDP) is a potent mechanism-based inhibitor of the enzyme RNR, which decomposes to an active alkylating furanone specie. The details of the inhibition mechanism are unknown, and experimental studies have indicated that some properties of the inactivation are dissimilar to those observed with a number of 2'-substituted 2'-deoxynucleotides mechanism-based inhibitors. To disclose the mechanism involved in RNR inactivation by CH2dCDP we explored the potential-energy surface in two different models of the system with different objectives in mind. In order to conveniently explore the reactional space, i.e. to study the possible reactions between the CH2dCDP and the RNR, we used a small model representing the active site of RNR with CH2dCDP using DFT. To provide further insights and efficiently account for the long-range RNR-CH2dCDP interactions and the stereochemical strain imposed by the protein scaffold we performed theoretical calculations on the more promising reactions using hybrid QM/MM calculations on a larger model system. We used quantum mechanics for the active-site region (CH2dCDP and active-site residues) and molecular mechanics for the surroundings (6373 atoms of the R1 monomer). The results obtained led us to understand the correct mechanism for RNR inactivation by CH2dCDP, and the furanone species formed presumably explains the dissimilarities observed with a number of 2'-substituted 2'-deoxynucleotides.
ABSTRACT
Nelfinavir (Viracept) is a potent, non-peptidic inhibitor of HIV-1 Protease, which has been marketed for the treatment of HIV infected patients. However, HIV-1 develops drug-resistance which decreases the affinity of Nelfinavir for the binding pocket of Protease. We present here three new variants of Nelfinavir, which we have designed with computational tools, with greater affinity for HIV-1 Protease than Nelfinavir itself. Accordingly, we have introduced rational modifications in Nelfinavir, optimizing its affinity to the most conserved amino acids in Protease, in order to increase the efficiency of the three new inhibitors. Minimization and molecular dynamics simulations have been carried out on four complexes, HIV-1 Protease with Nelfinavir and subsequently with the new inhibitors, respectively, in order to analyze the behavior of the systems. Additionally, we have calculated the binding free energy differences Protease:inhibitor, which gave us a quantitative idea of the new molecules inhibitory efficiency in silico.
