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Query: EC:3.4.23.16 (HIV-1 protease)
 2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A general scheme for obtaining a fluorescent donor/acceptor peptide substrate via solid-phase synthesis methodology is presented. The key feature of this method is the design of a glutamic acid derivative that has been modified on the carboxyl side chain with a 5-[(2'-aminoethyl)-amino]naphthelenesulfonic acid (EDANS) to create a fluorescent donor moiety that can be incorporated near the C-terminus of the peptide substrate. The corresponding fluorescent acceptor group containing a 4-[[4-(dimethylamino)phenyl]azo]benzoic acid (DABCYL) can then be attached to the resin-bound peptide at the N-terminus while all side-chain groups are still fully protected. Substrates for renin and HIV proteinase are synthesized as examples.
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PMID:A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis. 143 87

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.
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PMID:A side chain at position 48 of the human immunodeficiency virus type-1 protease flap provides an additional specificity determinant. 788 51

A series of 47 N-truncated reduced bond inhibitors, systematically modified at individual positions (P1, P'1, P'2, P'3, and P'4), were synthesized and used to map the subsite preferences of HIV-1 protease. The tight binding inhibitor of HIV-1 protease t-butoxycarbonyl-Phe-[CH2NH]Phe-Glu-Phe-NH2 (Ki = 0.2 nM) was chosen as the parent structure for further modifications. The P'2 glutamic acid was found to fit well into the S'2 subsite of the protease. The conformational restriction of any phenylalanine residue or saturation of more than one phenylalanine side chain in P'1 or P'3 lead is to a large Ki increase. Introduction of tyrosine in the P1 position improves the binding by an order of magnitude. The S'4 subsite of the protease was shown to accommodate large structural changes in the inhibitor at this position. Therefore P'4 may serve as an ideal region for further modification in order to improve bioavailability of this type of compound. An improved method of direct comparison of tight binding inhibitors with subnanomolar Ki values has been described.
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PMID:Specificity mapping of HIV-1 protease by reduced bond inhibitors. 832 74

The human immunodeficiency virus type-1 (HIV-1) encodes a protease which is essential for the production of infectious virus. The protease prefers substrates that contain glutamic acid or glutamine at the P2' position. The catalytic role of these residues has been studied by using a highly specific fluorogen substrate, 2-aminobenzoyl-Thr-Ile-Nle-Phe(NO2)-Gln-Arg (substrate QR), and its counterpart (substrate ER) containing Glu in place of Gln. The newly designed substrate ER that contains a pair of charged residues at P2' and P3' sites is the most specific substrate described so far for HIV-1 protease. The specificity rate constant (kcat/Km = 2.1 x 10(7) M-1 s-1) approaches, but does not reach, the diffusion limit. This follows from the appreciable solvent kinetic deuterium isotope effects on the rate constants, indicating that, independent of the salt concentration, the rate-limiting step of the catalysis is a chemical process rather than a physical one. The reaction also has positive entropy of activation. On the other hand, the rate-limiting step for substrate QR changes with increasing salt concentration from a physical to chemical step, while the negative activation entropy becomes positive. The rate increase with substrate ER is 50-fold with respect to substrate QR in the presence of 0.1 M NaCl and diminishes to 3.5-fold at 2.0 M NaCl concentration, as a consequence of a considerable rate increase at high salt concentration with substrate QR but not with substrate ER. The Km value is much lower for the substrate ER (0.8 microM) than for substrate QR (15 microM), indicating a more effective binding for substrate ER at 0.1 M NaCl. Unexpectedly, the strong binding appears to be achieved by the unionized form of Glu in P2', as follows from the remarkably different pH-rate profiles for substrates QR and ER. The effective binding elicited by the glutamic acid may be utilized in designing inhibitors for therapeutic purposes.
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PMID:Rate-determining steps in HIV-1 protease catalysis. The hydrolysis of the most specific substrate. 894 73

A bacteriophage T4-derived protein expression, packaging and processing system was used to create recombinant phage that encode, produce and package a protein composed of human HIV-1 protease fused to green fluorescent protein (GFP). The fusion protein is targeted within the phage capsid by an N-terminal capsid targeting sequence (CTS), which is cleaved through proteolysis by the viral scaffold protease P21. The fusion protein is designated CTS [symbol see text] GFP:PR. The [symbol see text] symbol indicates the linkage peptide sequence leu(ile)-N-glu that is cleaved by the T4 head morphogenetic proteinase gp21 during head maturation. The fusion protein is fluorescent and has protease activity as detected by the appearance of the expected substrate cleavage product on a Western blot. CTS [symbol see text] GFP:PR packaging occurs at about 200 molecules per phage particle. The CTS [symbol see text] GFP:PR fusion protein, when protected within the phage capsid, has been maintained stably for over 16 months at 4 degrees C. Production and storage of fusion protein within the phage circumvents problems of toxicity and solubility encountered with E. coli expression systems. Because recombinant phage inhibit host proteolytic enzymes, foreign proteins are stabilized. This phage system packages and processes the fusion protein by means of the CTS. Proteins can be purified from the phage to give high yields of soluble, proteolytically processed protein. The T4 phage packaging system provides a novel means of identification, purification and long-term storage of toxic proteins whose folding and DNA-directed activities can be studied readily in vivo.
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PMID:GFP:HIV-1 protease production and packaging with a T4 phage expression-packaging processing system. 986 54

This study reported the prevalence and pattern of viral replication-associated HIV-1 protease codon 35 amino acid insertions among treatment-naive patients in Hong Kong. The transmission and divergence date of these inserted strains was also investigated. The pol gene of 264 local HIV-1 isolates was sequenced and phylogenetic analysis was performed. The transmission history of protease codon 35-inserted HIV-1 strains in Hong Kong was estimated by the Bayesian coalescent method. This insertion was detected in 12 (4.55%) among 264 treatment-naive subtype B HIV-1 patients in Hong Kong, which was 20-times higher than the prevalence in the western countries. Among these strains, eight carried a glutamic acid (GAA) insertion (E35E_E), two carried an aspartic acid (GAC) insertion (E35E_D), and two carried a glycine (GGA) insertion (E35E_G). E35E_D and E35E_E insertions were the first to be reported. All the 12 inserted sequences clustered in the same lineage of the phylogenetic tree, indicating the possibility of transmission of this insertion. Epidemiological investigation revealed the major route of infection for this inserted strain in Hong Kong was associated mainly among homosexual Chinese males. The evolutionary rate of these inserted strains was similar to other subtype B HIV-1 strains. Through coalescent-based analysis, the divergence date of the protease codon 35-inserted strains in Hong Kong was 1995. Our findings demonstrate the epidemic pathways of viral fitness-related HIV-1 protease codon 35-inserted isolates in Hong Kong. The effect of these novel insertions on viral fitness and drug susceptibility requires further investigation.
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PMID:Molecular epidemiology and divergence of HIV type 1 protease codon 35 inserted strains among treatment-naive patients in Hong Kong. 1842 35

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-L-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.
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PMID:Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors. 2054 52