Resistant mechanism against nelfinavir of human immunodeficiency virus type 1 proteases

Inhibitors against human immunodeficiency virus type-1 (HIV-1) proteases are finely effective for anti-HIV-1 treatments. However, the therapeutic efficacy is reduced by the rapid emergence of inhibitor-resistant variants of the protease. Among patients who failed in the inhibitor nelfinavir (NFV) treatment, D30N, N88D, and L90M mutations of HIV-1 protease are often observed. Despite the serious clinical problem, it is not clear how these mutations, especially nonactive site mutations N88D and L90M, affect the affinity of NFV or why they cause the resistance to NFV. In this study, we executed molecular dynamics simulations of the NFV-bound proteases in the wild-type and D30N, N88D, D30N/N88D, and L90M mutants. Our simulations clarified the conformational change at the active site of the protease and the change of the affinity with NFV for all of these mutations, even though the 88th and 90th residues are not located in the NFV-bound cavity and not able to directly interact with NFV. D30N mutation causes the disappearance of the hydrogen bond between the m-phenol group of NFV and the 30th residue. N88D mutation alters the active site conformation slightly and induces a favorable hydrophobic contact. L90M mutation dramatically changes the conformation at the flap region and leads to an unfavorable distortion of the binding pocket of the protease, although 90M is largely far apart from the flap region. Furthermore, the changes of binding energies of the mutants from the wild-type protease are shown to be correlated with the mutant resistivity previously reported by the phenotypic experiments.     

Antiviral activity of polyoxomolybdoeuropate PM-104 against human immunodeficiency virus type 1

A polyoxomolybdoeuropate PM-104 (NH4)12H2[Eu4(MoO4)(H2O)16(Mo7O24)4].13H2O was found to be a potent inhibitor of the growth of human immunodeficiency virus type 1 (HIV-1), a causative agent of acquired immunodeficiency syndrome (AIDS). On the basis of TI50 [median cytotoxic concentration (CC50)/median effective concentration (EC50)], the in vitro anti-HIV-1 activity of PM-104 is favorably comparable to that of a heteropolyoxotungstate PM-19 K7[PTi2W10O40].6H2O, which is one of the most potent HIV-1 inhibitors among the polyoxometalates so far tested. The heteropolyoxomolybdate with a potent anti-HIV-1 activity is introduced for the first time in this communication.     

Antiviral activities of glycyrrhizin and its modified compounds against human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 1 (HSV-1) in vitro.

Chemically modified compounds of glycyrrhizin have been synthesized and evaluated for their inhibitory effect on the replication of human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 1 (HSV-1). Among them, the 11-deoxo compound having a heteroannular diene structure at the C and D rings proved as active against HIV-1 as glycyrrhizin in MT-4 and MOLT-4 cells. It completely inhibited HIV-1-induced cytopathogenicity in both cell lines at a concentration of 0.16 mM. The compound was also effective against HSV-1 with a 50% inhibitory concentration of 0.5 mM [corrected].     

Unraveling the concerted catalytic mechanism of the human immunodeficiency virus type 1 (HIV-1) protease: a hybrid QM/MM study

Structural Chemistry - We give an account of a one-step concerted catalytic mechanism of HIV-1 protease (PR) hydrolysis of its natural substrate using a hybrid QM/MM method. The mechanism is a...     

Capillary electrophoretic separation of protease inhibitors used in human immunodeficiency virus therapy

A new capillary electrophoretic method for trace analysis of gamma-cyclodextrin, gamma-CD, in a sample of beta-CD has been developed, building on our recent work in which the tetraphenylborate ion, Ph4B-, was found to bind to gamma-CD three orders of magnitude more strongly than to beta-CD. The method involves measurement of the change of net electrophoretic mobility of Ph4B- and its CD complexes in a background electrolyte containing a fixed concentration of beta-CD. Good linearity was found between 1/deltamu and 1/Cgamma where deltamu is the difference in the mobility of Ph4B- in the beta-CD solution at a given and at zero concentration of gamma-CD, and Cgamma the gamma-CD concentration. The limit of detection for gamma-CD in a beta-CD sample was found to be 0.020% (w/w), and high precision and accuracy were obtained.     

Inhibitory effects on HIV-1 protease of tri-p-coumaroylspermidine from Artemisia caruifolia and related amides.

From a methanol extract of Artemisia caruifolia, which showed a moderate inhibitory activity on HIV-1 protease in a preliminary screening, N1,N5,N10-tri-p-coumaroylspermidine and three dicaffeoylquinic acids were isolated. The former compound was found to appreciably inhibit HIV-1 protease. Of related amides which were chemically synthesized, N1,N5,N10,N14-tetra-p-coumaroylspermine and N1,N4,N7,N10,N13-penta-p-coumaroylte-traethylenepentamine inhibited HIV-1 protease more potently than N1,N5,N10-tri-p-coumaroylspermidine.     

Inhibitory effects of constituents from Cynomorium songaricum and related triterpene derivatives on HIV-1 protease

From CH2Cl2 and MeOH extracts of the stems of Cynomorium songaricum RUPR. (Cynomoriaceae), ursolic acid and its hydrogen malonate were isolated as inhibitors of human immunodeficiency virus type 1 (HIV-1) protease, with 50% inhibitory concentrations (IC50) of 8 and 6 microM, respectively. Amongst various synthesized dicarboxylic acid hemiesters of related triterpenes, inhibitory activity tended to increase in the order of oxalyl, malonyl, succinyl and glutaryl hemiesters, for triterpenes such as ursolic acid, oleanolic acid and betulinic acid. The most potent inhibition was observed for the glutaryl hemiesters, with an IC50 of 4 microM. From the water extract of the stems of C. songaricum, flavan-3-ol polymers, consisting of epicatechin as their extender flavan units, were also found to be potent inhibitory principles against HIV-1 protease.     

Synthesis and biological evaluation of novel isopropanolamine derivatives as non-peptide human immunodeficiency virus protease inhibitors

Novel potential human immunodeficiency virus (HIV) protease inhibitors were designed by a combination of nelfinavir and amprenavir motifs. The designed compounds were prepared by a facile synthetic route and their stereochemistry was further confirmed by a stereospecific synthesis from commercially available (S)-2-oxiranylmethyl m-nitrobenzenesulfonate. All compounds were tested for their ability in inhibiting HIV type 1 protease activity with the published method of reference 19. Derivatives 1a--u exhibited moderate to significant inhibitory activities in preliminary bioassay. The best compound 1a has IC50 value of 0.02 microM, comparable to that of amprenavir. A docking study on compounds 1a--u was performed using the published X-ray crystal structure of HIV type 1 protease, all compounds bound to the HIV type 1 protease in an extended conformation and the scaffoldings of the binding conformations could be aligned quite well. Comparative molecular field analysis (CoMFA) study was performed to explore the specific contributions of electrostatic and steric effects in the binding of these new compounds to HIV type 1 protease and a predictive CoMFA model was built with thirteen compounds as training set. Test analysis of other five compounds as test set demonstrated that the CoMFA model has strong predictive ability to this series of compounds. It will be very useful to further optimize the designed inhibitors.     

Ultratrace voltammetric method for the detection of DNA sequence related to human immunodeficiency virus type 1

An ultra-trace voltammetric method was developed for the determination of single strand DNA (ss-DNA) related to the human immunodeficiency virus type 1 (HIV-1). It is based on the signal amplification of carbon nanotubes loaded with silver nanoparticles and placed on a gold microelectrode. The capture ss-DNA (a 21-mer) possessing a thiol group at the 3?? end was self-assembled onto the surface of the gold microelectrode. It was then hybridized with target HIV-1 ss-DNA (a 42-mer) and further hybridized with the electrochemical probe (a 18-mer ss-DNA) tagged with multiwall carbon nanotubes and loaded with silver nanoparticles. The resulting formation of a DNA sandwich conjugate led to a strong electrochemical oxidation signal that was linearly proportional to the concentration of HIV-1 ss-DNA in the range from 1.0 to 100?pM. The detection limit was 0.5?pM (at an S/N of 3). This was equivalent to 0.05?fmol of HIV-1 ss-DNA in a volume of 20???L. The relative standard deviation was 4.0% at 1.0?pM (n?=?11). Non-complementary ss-DNA of HIV-1 ss-DNA was effectively discriminated. This work demonstrates that the employment of the microelectrode and a sandwich hybridization model is promising in terms of sensitive and selective electrochemical detection of DNA.

12-O-acetylphorbol-13-decanoate potently inhibits cytopathic effects of human immunodeficiency virus type 1 (HIV-1), without activation of protein kinase C.

Through bioactivity-guided fractionation, eight phorbol diesters, including five new ones (1-5), were isolated from the seeds of Croton tiglium collected in Egypt. 12-O-Acetylphorbol-13-decanoate (6) and 12-O-decanoylphorbol-13-(2-methylbutyrate) (4) potently inhibited the HIV-1-induced cytopathic effect on MT-4 cells (IC100 values of 7.6 ng/ml and 7.81 micrograms/ml, and CC0 values of 62.5 micrograms/ml and 31.3 micrograms/ml, respectively) without activating protein kinase C.     

Two new triterpenes from the Rhizome of Dryopteris crassirhizoma, and inhibitory activities of its constituents on human immunodeficiency virus-1 protease

Two new hopane type triterpenes, named dryopteric acids A (1) and B (2), were isolated from the Rhizome of Dryopteris crassirhizoma (Aspiadaceae) together with sixteen known compounds (3-18). Of isolated compounds, ursolic acid (15), and dryopteric acid A (1) and B (2) showed potent inhibitory activities against HIV-1 protease with IC50 values of 8.9-44.5 microM. In addition, acetylated compounds 1 and 2 appreciably increased inhibitory activities with their IC50 values of 1.7 and 10.8 microM, respectively.     

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.     

Characterizing the protonation states of the catalytic residues in apo and substrate-bound human T-cell leukemia virus type 1 protease

Human T-cell leukemia virus type 1 (HTLV-1) protease is an attractive target when developing inhibitors to treat HTLV-1 associated diseases. To study the catalytic mechanism and design novel HTLV-1 protease inhibitors, the protonation states of the two catalytic aspartic acid residues must be determined. Free energy simulations have been conducted to study the proton transfer reaction between the catalytic residues of HTLV-1 protease using a combined quantum mechanical and molecular mechanical (QM/MM) molecular dynamics simulation. The free energy profiles for the reaction in the apo-enzyme and in an enzyme – substrate complex have been obtained. In the apo-enzyme, the two catalytic residues are chemically equivalent and are expected to be both unprotonated. Upon substrate binding, the catalytic residues of HTLV-1 protease evolve to a singly protonated state, in which the OD1 of Asp32 is protonated and forms a hydrogen bond with the OD1 of Asp32′, which is unprotonated. The HTLV-1 protease–substrate complex structure obtained from this simulation can serve as the Michaelis complex structure for further mechanistic studies of HTLV-1 protease while providing a receptor structure with the correct protonation states for the active site residues toward the design of novel HTLV-1 protease inhibitors through virtual screening.     

Molecular modeling studies of human immunodeficiency virus type 1 protease inhibitors using three‐dimensional quantitative structure‐activity relationship,virtual screening,and docking simulations

In this paper, two 3‐dimensional quantitative structure‐activity relationship models for 60 human immunodeficiency virus (HIV)‐1 protease inhibitors were established using random sampling analysis on molecular surface and translocation comparative molecular field vector analysis (Topomer CoMFA). The non–cross‐validation (r²), cross‐validation (q²), correlation coefficient of external validation (Q²_ext), and F of 2 models were 0.94, 0.80, 0.79, and 198.84 and 0.94, 0.72, 0.75, and 208.53, respectively. The results indicated that 2 models were reasonable and had good prediction ability. Topomer Search was used to search R groups in the ZINC database, 20 new compounds were designed, and the Topomer CoMFA model was used to predicate the biological activity. The results showed that 18 new compounds were more active than the template molecule. So the Topomer Search is effective in screening and can guide the design of new HIV/AIDS drugs. The mechanism of action was studied by molecular docking, and it showed that the protease inhibitors and Ile50, Asp25, and Arg8 sites of HIV‐1 protease have interactions. These results have provided an insight for the design of new potent inhibitors of HIV‐1 protease.