Could angiotensin I be produced from a renin substrate by the HIV-1 protease?

The standard angiotensin I (Ang I) radioimmunoassay for renin activity determination is a useful clinical tool for the diagnosis of high renin levels in certain cases of hypertension. It depends upon the liberation of Ang I from human plasma angiotensinogen. We considered whether a commercially available synthetic tetradecapeptide (TDP), Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, would produce authentic Ang I upon incubation with protease from human immunodeficiency virus type 1 (HIV-1). This peptide is also known to be cleaved by renin at the Leu-Leu bond to yield the decapeptide Ang I. When the TDP is incubated with the HIV-1 protease, the peptide is readily hydrolyzed. Product formation is linear with respect to time and enzyme concentration. HPLC analysis of reaction products showed two new peaks, as one would expect from the cleavage of a TDP into a decapeptide and a tetrapeptide. Amino acid analysis of HPLC-purified peaks confirmed that the HIV-1 protease cleaves TDP at the Leu10-Leu11 site to produce the desired decapeptide, Ang I. Production of Ang I by the HIV-1 protease, like human renin, is inhibited in the presence of a protease inhibitor. Implications of the discovery of an HIV-1 protease substrate that produces authentic Ang I are discussed in light of a screening assay for soluble HIV-1 protease inhibitors.     

An ultrasensitive human immunodeficiency virus type 1 protease radioimmuno rate assay with a potential for monitoring blood levels of protease inhibitors in acquired immunodeficiency disease syndrome patients.

The angiotensin I-based peptide Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser yields angiotensin I (Ang I) and Leu-Glu-Glu-Ser upon hydrolysis by the human immunodeficiency virus type 1 (HIV-1) protease, but not by human renin. N-terminal sequencing of the reaction products showed that the HIV-1 protease cleaved exclusively at the Leu-Leu bond. The rate of Ang I formation can be measured by a radioimmunoassay, since the parent peptide has minimal cross reactivity in this assay. The rate of enzymatic hydrolysis is maximal at pH 4.5-5.0 and at an ionic strength of 1 M. At 37 degrees C, 0.1 M Na acetate buffer, pH 5.0, 1 M NaCl, 10% glycerol, 5% ethylene glycol, 1 mg/ml bovine serum albumin, and 3 mM EDTA, the reaction obeys Michaelis-Menten type kinetics with Km = 17.2 +/- 3.5 microM and kcat = 2.30 +/- 0.33 min-1. The activity assay readily quantitates as little as 0.25 nM of HIV-1 protease. The production of Ang I by the HIV-1 protease is inhibited in the presence of a HIV-1 protease inhibitor. The newly discovered substrate is relatively insensitive to human or monkey serum. Therefore, the effect of sera from 20 patients with advanced acquired immunodeficiency disease syndrome (AIDS) on Ang I production in the above assay system was examined. Results of this study indicate that it may be possible to adapt the above Ang I-based system to determine blood levels of HIV-1 protease inhibitors in AIDS patients during clinical trials.     

Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors.

A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible.     

Ultramicroassay for plasma renin concentration in the rat using the antibody-trapping technique

Using the antibody-trapping technique, picogram quantities of angiotensin-I generated during 24 hr of incubation at 37°C were stable and fully protected against peptidases. The method employs purification of angiotensin-I antisera on DEAE-cellulose and purification of renin substrate by affinity chromatography using specific antirenin antibodies in order to remove endogenous renin. The assay was performed in a single tube without a transfer step in a total volume of 30 μl at pH 6,5 with incubation for 24 hr at 37°C. With a normal rat plasma renin concentration of 5 × 10^?4 GU ml^?1, the detection limit was 10 nl or a total of 5 × 10^?9 GU. In the range 20–125 nl, precision was ±10%.     

Substrate analog inhibitors of HIV-1 protease containing phenylnorstatine as a transition state element

Substrates of HIV-1 protease are classified into three groups (A, B and C) based on the amino acid residues present at P1' and P2' sites. Replacement of the scissile amide bond by phenylnorstatine in representative substrate analog sequences from class A, B and C, yielded inhibitors of HIV-1 protease. Of the twelve inhibitors synthesized in this series, class C substrate analog inhibitors are more potent inhibitors (Ki's 3.3-24 microM) than either class A or class B inhibitors. In this series of inhibitors, the (2S,3S) isomer of phenylnorstatine is preferred over the other isomers as a "transition state element" for design of inhibitors of HIV-1 protease.     

An improved method for measuring angiotensin I converting enzyme activity using a highly sensitive angiotensin II radioimmunoassay

A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmunoassay. To protect against angiotensin II destruction, bestatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His-Leu. The generation of as little as 12 pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radioimmunoassay for angiotensin II.     

A radiometric assay for HIV-1 protease

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.     

A cleavage enzyme-cytometric bead array provides biochemical profiling of resistance mutations in HIV-1 Gag and protease

Most protease-substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease-substrate cleavage assay, termed the cleavage enzyme- cytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescence utilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (k(cat)/K(M)) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme-substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.     

Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule inhibitors of protease dimerization

Dimerization of HIV-1 protease subunits is essential for its proteolytic activity, which plays a critical role in HIV-1 replication. Hence, the inhibition of protease dimerization represents a unique target for potential intervention of HIV-1. We developed an intermolecular fluorescence resonance energy transfer-based HIV-1-expression assay employing cyan and yellow fluorescent protein-tagged protease monomers. Using this assay, we identified non-peptidyl small molecule inhibitors of protease dimerization. These inhibitors, including darunavir and two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 microm and blocked HIV-1 replication with IC(50) values of 0.0002-0.48 microm. These agents also inhibited the proteolytic activity of mature protease. Other approved anti-HIV-1 agents examined except tipranavir, a CCR5 inhibitor, and soluble CD4 failed to block the dimerization event. Once protease monomers dimerize to become mature protease, mature protease is not dissociated by this dimerization inhibition mechanism, suggesting that these agents block dimerization at the nascent stage of protease maturation. The proteolytic activity of mature protease that managed to undergo dimerization despite the presence of these agents is likely to be inhibited by the same agents acting as conventional protease inhibitors. Such a dual inhibition mechanism should lead to highly potent inhibition of HIV-1.     

Renin activity determination using human plasma as a substrate

A method for human renin activity determination using human plasma as the substrate is described. Angiotensin I is generated by incubating renin with human plasma, which is available along with the commercial radioimmunoassay kit for angiotensin I. The method obviates the need to isolate and purify the substrate, human angiotensinogen, from human plasma. In addition, the assay is highly renin specific, sensitive, and convenient to use for the routine determination of active human renin during its isolation and purification from tissue extracts or from genetically engineered bacterial and nonbacterial expression systems.     

Comparative analysis of the sequences and structures of HIV-1 and HIV-2 proteases

The different isolates available for HIV-1 and HIV-2 were compared for the region of the protease (PR) sequence, and the variations in amino acids were analyzed with respect to the crystal structure of HIV-1 PR with inhibitor. Based on the extensive homology (39 identical out of 99 residues), models were built of the HIV-2 PR complexed with two different aspartic protease inhibitors, acetylpepstatin and a renin inhibitor, H-261. Comparison of the HIV-1 PR crystal structure and the HIV-2 PR model structure and the analysis of the changes found in different isolates showed that correlated substitutions occur in the hydrophobic interior of the molecule and at surface residues involved in ionic or hydrogen bond interactions. The substrate binding residues of HIV-1 and HIV-2 PRs show conservative substitutions of four residues. The difference in affinity of HIV-1 and HIV-2 PRs for the two inhibitors appears to be due in part to the change of Val 32 in HIV-1 PR to Ile in HIV-2 PR.     

Properties of renin substrate in rabbit plasma with a note on its assay

1. Rabbit plasma enzymes that degrade angiotensin I are inhibited completely by the combination of 2,3-dimercaptopropan-1-ol (10mm), EDTA (10mm) and chlorhexidine gluconate (0.005%, w/v). These compounds do not modify the reaction of renin with renin substrate and are termed the selective inhibitors. 2. The renin substrate concentration of plasma can be measured as angiotensin I content by incubating plasma plus the selective inhibitors with renin for a time sufficient to allow complete utilization of renin substrate. 3. This reaction obeys first-order kinetics to substrate concentrations of at least 1000ng. of angiotensin I content/ml. In general, the renin substrate concentrations of normal rabbit plasmas are less than 1000ng. of angiotensin I content/ml. Thus the time required for the complete release of angiotensin I from normal plasma is inversely related to renin activity and is independent of renin substrate concentration. 4. A method for the assay of renin substrate, taking these reaction kinetics into account, is presented.     

Proteases and their inhibitors: today and tomorrow.

A major incentive in inhibitor research is that control of limited proteolysis constitutes a valuable pharmacological tool. Protease inhibitors have proved to be successful in influencing pathogenesis in many experimental models but a breakthrough to use in human therapy has mainly been restricted to aprotinin and angiotensin converting enzyme (ACE) inhibitors. However, the success of ACE inhibitors as pharmacological tools in hypertension has proved to be a strong stimulant for new protease inhibitor approaches to drug therapy. While emphasis in the search for next generations of ACE inhibitors may move from the circulation renin-angiotensin system to the local tissue systems, including heart, brain and genital tract, persistent and insightful design of renin inhibitors has already yielded highly specific molecules with potent activities in several in vivo models. The development of orally effective long-acting inhibitors will finally allow an evaluation to be made of their therapeutic profile with regard to the family of ACE inhibitors. The close relationship between renin and HIV-1 protease presents an exceptional opportunity for transfer of the knowledge acquired in renin inhibitor development during the past decade, to an accelerated generation of specific HIV-1 protease inhibitors as effective agents in treatment of AIDS. The self-assembly of 2 identical monomers into a symmetrical structure in HIV-1 protease is not only an elegant way to create an active enzyme while encoding a minimal amount of genetic information, but is also in concordance with the bilobular active-site found in mammalian aspartic proteases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.     

Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin

Detection of proteolytic bond cleavage was achieved by taking advantage of the bioluminescence emission generated by the photoprotein aequorin. A genetically engineered HIV-1 protease substrate was coupled with a cysteine-free mutant of aequorin by employing the polymerase chain reaction to produce a fusion protein that incorporates an optimum natural protease cleavage site. The fusion protein was immobilized on a solid phase and employed as the substrate for the HIV-1 protease. Proteolytic bond cleavage was detected by a decrease in the bioluminescence generated by the aequorin fusion protein on the solid phase. A dose-response curve for HIV-1 protease was constructed by relating the decrease in bioluminescence signal with varying amounts of the protease. The system was also used to evaluate two competitive and one noncompetitive inhibitor of the HIV-1 protease. Among the advantages of this assay is that by using recombinant methods a complete bioluminescently labeled protease recognition site can be designed and produced. The assay yields very sensitive detection limits, which are inherent to bioluminescence-based methods. An application of this system may be in the high-throughput screening of biopharmaceutical drugs that are potential inhibitors of a target protease.     

Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

The success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. In addition, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.     

Renin substrate (angiotensinogen) preparations in the determination of prorenin and renin: evidence for extrarenal plasma prorenin and its renal "convertase"

Standard methods for determining prorenin-renin concentrations in plasma (PRC) and other tissues require the addition of exogenous renin substrate (angiotensinogen) to improve the kinetics of the renin reaction. We studied the effects of substrate prepared from normal human plasma fraction Cohn IV-4, or from nephrectomized (2NX) sheep plasma, on PRC of normal and 2NX human plasmas before and after prorenin activation by acid, cold, and trypsin, and compared the results with plasma renin activities (PRA, no added substrate). Plasmas from 2NX men exhibited negligible basal PRA, indicating that very little, if any, renin had been formed from the extrarenal prorenin they contained, and suggesting the lack of an endogenous prorenin activating mechanism, or "convertase," of probable renal origin. Prorenin was demonstrable by tryptic activation, more than by acid or cold, at up to about 30% of normal. Addition of Cohn IV-4 substrate to 2NX plasma unexpectedly produced (i) a basal PRC value higher than in normal plasma, (ii) total renin values after activation by acid, cold, and trypsin that were much closer to normal values than reflected by PRA methodology, without a commensurate increase (if anything a decrease) in prorenin as a percentage of total renin estimated by all activation methods, and (iii) substantial equalization of activation effects such that trypsin was no longer more effective than acid and cold (and this was also noted with normal plasma). The skewing effect of adding Cohn IV-4 substrate on the PRC of 2NX plasma was much greater than in normal plasma, even though 2NX plasma already had an above normal level of endogenous substrate and should have been influenced less. Enhancement of PRC was very pronounced even when Cohn IV-4 was added to make up only 9% of total (endogenous + exogenous) substrate in the incubation system, suggesting that it was not the added substrate but a renin-generating contaminant that inflated the PRC. Such inflation could be blocked by adding protease inhibitors, suggesting that the responsible protease(s) acted as a prorenin "convertase" that generated new renin from renal and (or) extrarenal prorenin contributed by the added substrate, as well as by the plasma being assayed. One component of convertase could be kallikrein, which was identified by chromogenic assay, the importance of which relative to total convertase activity is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)     

Peptide inhibitors of converting enzyme.

Human plasma and atypical lung converting enzyme, and porcine plasma converting enzyme are substantially inhibited by other components of the renin-angiotensin system, and by angiotensin II and its analogues. Des-Asp¹ angiotensin II (angiotensin III) 0.1 mM and tridecapeptide renin substrate 0.1 mM are both effective inhibitors of human lung, plasma and porcine plasma converting enzymes. Des-Asp¹-Arg² angiotensin II also was an effective inhibitor of plasma enzymes. Bradykininase activity (kininase II) of the converting enzymes was also inhibited by angiotensin I, angiotensin III, tetradecapeptide renin substrate and tridecapeptide renin substrate. The substantial kininase and converting enzyme inhibitory effects of components of the renin-angiotensin system, suggest a potential close physiologic relationship between the kallikrein-kinin system and the renin-angiotensin system.