Characterization of Alzheimer's beta -secretase protein BACE. A pepsin family member with unusual properties.

The cerebral deposition of amyloid beta-peptide is an early and critical feature of Alzheimer's disease. Amyloid beta-peptide is released from the amyloid precursor protein by the sequential action of two proteases, beta-secretase and gamma-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of beta-secretase. Here we demonstrate that BACE is an N-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a secreted Fc fusion-form of BACE (BACE-IgG) that contains the entire ectodomain for a detailed analysis of posttranslational modifications. This molecule starts at Glu(46) and contains four N-glycosylation sites (Asn(153), Asn(172), Asn(223), and Asn(354)). The six Cys residues in the ectodomain form three intramolecular disulfide linkages (Cys(216)-Cys(420), Cys(278)-Cys(443), and Cys(330)-Cys(380)). Despite the conservation of the active site residues and the 30-37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member.

The intermolecular disulfide bridge of human glial cell line-derived neurotrophic factor: its selective reduction and biological activity of the modified protein.

Recombinant human glial cell line-derived neurotrophic factor has been implicated to have therapeutic potential in the treatment of neurodegenerative diseases. The mature protein is a single polypeptide of 134 amino acid residues and functions as a disulfide-linked dimer. Reduction of the protein with dithiothreitol at pH 7.0 and in the absence of denaturant showed that the single intermolecular cystine bridge was reduced preferentially. Direct alkylation of the generated free sulfhydryl group using iodoacetamide or iodoacetate without denaturant was incomplete. Unfolding the protein in 6 M guanidine hydrochloride prior to the modification showed rapid disulfide scrambling. However, the sulfhydryl-modifying reagent N-ethylmaleimide was able to label quantitatively the free cysteinyl residue in the absence of any added chaotropic agent. By a combination of peptide mapping, Edman degradation, and mass spectrometric analysis, the labeled residue was identified to be Cys101, hence verifying the location of the intermolecular disulfide bond. The modified protein behaved as a noncovalent dimer when chromatographed through a Superdex 75 column under nondenaturing conditions and was comparable in biological activity to an unmodified control sample. The results therefore indicate that the intermolecular disulfide bridge of the protein is not essential for its biological function.

Identification of Asp95 as the site of succinimide formation in recombinant human glial cell line-derived neurotrophic factor.

Human glial cell line-derived neurotrophic factor is a single polypeptide of 134 amino acids and functions as a disulfide-linked dimer. Incubation of the protein in pH 5.0 and at 37 degreesC for 1 week showed that 5% of the material was converted to a form that eluted after the major protein peak on a cation-exchange column. The modified component gave an average molecular mass of 30367.0 u (theoretical = 30384.8 u). Within measurement error, this 17.8-u decrease in mass indicated the loss of a water molecule. This observation, together with the protein's behavior on cation-exchange chromatography and the mode of incubation used to generate the modification, was consistent with cyclic imide (succinimide) formation at an aspartyl residue. Hence, only a monomer of the dimeric protein was modified. The modified monomer was purified and subjected to peptic degradation. By a combination of N-terminal analysis and mass spectrometry, the region containing Asp95-Lys96 was identified to be modified. This was further confirmed by carboxypeptidase Y digestion of the modified peptide where the modified region was found to be resistant to further enzymatic degradation. Furthermore, incubation of the modified monomer in pH 8. 5 for 2 h yielded two peaks, in agreement with the succinimide model where the cyclic imide was hydrolyzed into a mixture of isoaspartate and aspartate. Tryptic mapping of the isoaspartyl-containing protein showed that Asp95 was refractory to Edman degradation, confirming it was in the isoaspartate form. Hence, the modification observed was due to succinimide formation at Asp95. This is the first report of succinimide formation at an Asp-Lys linkage.

Human leptin receptor. Determination of disulfide structure and N-glycosylation sites of the extracellular domain.

The leptin receptor (OB-R) is a member of the class I cytokine receptor family and mediates the weight regulatory effects of its ligand through interaction with cytoplasmic kinases. The extracellular domain of this receptor is comprised of two immunoglobulin-like and cytokine-receptor homology domains each and type III fibronectin domains. The extracellular domain of human leptin receptor was expressed in and purified from Chinese hamster ovary cells and was found to contain extensive N-glycosylation (approximately 36% of the total protein). The purified protein had a molecular weight of approximately 145,000 and exhibited ligand binding ability as evidenced by formation of ligand-receptor complex, followed by chemical cross-linking. The determined disulfide motif of the soluble leptin receptor contained several distinct cystine knots as well as 10 free cysteines. The N-glycosylation analysis revealed that Asn624 of the WSXWS motif (residues 622-626) within the C-terminal cytokine receptor homology domain was glycosylated, indicating that this region is solvent-exposed. On the other hand, the N-terminal WSXWS motif was not glycosylated.

Determination of disulfide structure in agouti-related protein (AGRP) by stepwise reduction and alkylation.

The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical methods using partial reduction with tris(2-carboxyethyl)phosphine under acidic conditions, followed by direct alkylation with N-ethylmaleimide or fluorescein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible disulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C88, C67-C82, and C81-C99, respectively. The chemically defined disulfide connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109.

Human neurotrophin-3: a one-step peptide mapping method and complete disulfide characterization of the recombinant protein.

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an initial denaturation of NT-3 in 6 M guanidinium chloride (pH6) for 2 hr at 37 degrees C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors.

Analysis of translational termination of recombinant human methionyl-neurotrophin 3 in Escherichia coli.

A highly efficient UGA stop codon readthrough event during the synthesis of human neurotrophin 3 in E. coli is described. The incorporation of a Trp residue at the UGA stop codon is confirmed combining both the chemical analyses and the molecular and genetic data in this report. The 3' adjacent nuleotide to the UGA stop codon plays a crucial role in determining the readthrough efficiency in the order of A > G > C > U. The replacement of UGA with UAA or UAG totally abolished this readthrough phenomenon and the use of StpR host cells also prevented the occurrence of UGA readthrough. Gene dosage (or plasmid copy number) effect was not indicated in this event; however, the titration of RF-2 by mRNA transcripts under over-expression conditions might explain why tRNAtrp competes so well with RF-2 for UGA. Another apparently less produced readthrough product resulting from a transcript with no stop codon is also recorded, and the addition of a second in-frame stop codon increased the amount of the observed readthrough product.

Evaluation of a vitamin-cloaking strategy for oligopeptide therapeutics: biotinylated HIV-1 protease inhibitors.

The outstanding limitations to the oligopeptide as a therapeutic agent are poor oral availability and rapid biliary clearance. To address these concerns a series of eight peptidic HIV-1 protease inhibitors containing the structural segment of the vitamin biotin have been prepared. These have been evaluated with regard to the hypothesis that this vitamin would cloak the peptidic character of these oligopeptides, and thus impart to these inhibitors the potential for absorption and distribution via biotin transporters and receptors. By iterative optimization about a -Cha psi[CH-(OH)CH(OH)]Val- core inhibitory insert, three particularly potent inhibitors (K(i) < or = 10 nM) of the HIV-1 protease were obtained. Although excellent cell culture antiviral activity is observed for other peptidic protease inhibitors of comparable affinity, none in this series exhibited satisfactory antiviral activity. This failure is attributed to the incompatibility of the hydrophilic and hydrogen-bonding biotin segment, with the facile membrane permeability and intracellular access presumably required for antiviral activity. The ability of the biotin to cloak the peptide, and thus render the overall appearance of the conjugate as that of a vitamin, was evaluated. Four of this series were evaluated for recognition by the Caco-2 cell intestinal biotin transporter. None inhibited competitively biotin uptake, indicating a lack of recognition. A vitamin may bind to a specific protein carrier, and thus attain an improved serum profile (by resistance to biliary clearance) and advantageous delivery to cells. Therefore, the serum concentrations of three were evaluated following an iv bolus in a rat model for serum clearance. One of the three protease inhibitors (L-idonamide, 6-cyclohexyl-2,5,6-trideoxy-2-(1-methylethyl)-5-[[3-methyl-1-oxo-2-[[5- (hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl)-1- oxopentyl]amino]butyl]amino]-N-[2-methyl-1-[[(2- pyridinylmethyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta (1R*,2R*,3R*),6 alpha]]-) sustained a more than 5-fold increase in serum concentration at all time points relative to the benchmark structure. The remaining two had serum concentrations at least equal to the benchmark, suggestive of improved resistance to clearance. One (L-idonamide,6-cyclohexyl-2,5,6-trideoxy-5-[[2-[[5-(hexahydro-2-ox o-1H- thieno-[3,4-d]imidazol-4-yl)pentyl]thio]benzoyl]amino]-2-(1- methylethyl)-N-[2-methyl-1-[[(2-pyridinyl- methyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta(1R*,2R*),6a alpha]]-) was prepared as a complex with the biotin-binding protein avidin. Avidin may resemble an endogenous serum biotin carrier protein.(ABSTRACT TRUNCATED AT 400 WORDS)

The crystallographic structure of the protease from human immunodeficiency virus type 2 with two synthetic peptidic transition state analog inhibitors.

The crystal structure of human immunodeficiency virus (HIV) type 2 protease has been determined in complexes with peptidic inhibitors Noa-His-Cha psi [CH(OH)CH(OH)]Val-Ile-Amp (U75875) and Qnc-Asn-Cha psi [CH(OH)CH2]Val-Npt(U92163) (where Noa is naphthyloxyacetyl, Cha is cyclohexylalanine, Amp is 2-aminomethylpyridine, Qnc is quinoline-2-carbonyl, and Npt is neopentylamine), which have dihydroxyethylene and hydroxyethylene moieties, respectively, in place of the normal scissile bond of the natural ligand. The complexes crystallize in space group P2(1)2(1)2(1), with one dimer-inhibitor complex per asymmetric unit and average cell dimensions of a = 33.28 A, b = 45.35 A, c = 135.84 A. Data were collected to approximately 2.5-A resolution. The model structures were refined with resulting R-factors of around 0.19. As expected, the HIV-2 protease structure is approximately C2-symmetric with a gross structure very similar to that of the HIV-1 enzyme. The inhibitors bind in an extended conformation positioned lengthwise in the binding cleft in a manner similar to that found in the HIV-1 protease-inhibitor complexes previously reported. The substitution of the bulkier Ile82 side chain in the HIV-2 protease may help explain the better ability of HIV-2 protease to bind and hydrolyze ligands with small P1 and P1' side groups. It appears that differences in specificity between the proteases of HIV-1 and HIV-2 are not merely a result of simple side chain substitutions, but may be complicated by differences in main chain flexibility as well.

Large scale purification and refolding of HIV-1 protease from Escherichia coli inclusion bodies.

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered in Escherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-1 protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band of M(r) approximately 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.

Inhibitors of the protease from human immunodeficiency virus: synthesis, enzyme inhibition, and antiviral activity of a series of compounds containing the dihydroxyethylene transition-state isostere.

A number of potential HIV protease inhibitory peptides that contain the dihydroxyethylene isostere were prepared and evaluated for their enzyme binding affinity and antiviral activity in cell cultures. From the template of a previously reported active peptide A, modifications at the N- and C-terminal groups were assessed for potential maintenance of good inhibitory activity of the resulting peptides. Among the active peptides found, peptide XVIII exhibited potent enzyme inhibitory activity. Interestingly, the previously reported, effective 1(S)-amino-2(R)-hydroxyindan C-terminal group for the preparation of very active HIV protease inhibitory peptides could not be applied to the template of peptide XVIII. Molecular modeling of peptide XVIII was studied using the X-ray crystal structure of peptide A as a starting point in order to study the likely conformation of peptide XVIII in the active-site cleft. Relative binding conformations of peptide A and XVIII were obtained, although the reason for poor binding affinity for a number of congeneric peptides in this report was not straightforwardly apparent. More importantly, however, peptide XVIII was found to exhibit more effective antiviral activity in the HIV-1/PBMC assay than the reference peptide A which was previously reported to be approximately equal in efficacy to the reverse transcriptase inhibitor AZT in this assay.

HIV protease (HIV PR) inhibitor structure-activity-selectivity, and active site molecular modeling of high affinity Leu [CH(OH)CH2]Val modified viral and nonviral substrate analogs.

This report details the structure-activity relationships of the HIV gag substrate analog Val-Ser-Gln-Asn-Leu psi[CH(OH)CH2]Val-Ile-Val (U-85548E), an inhibitor exhibiting subnanomolar affinity towards HIV type-1 aspartic proteinase (HIV-1 PR). Our data show that the P1-P2' tripeptidyl sequence provides the minimal chemical determinant for HIV-1 PR binding. We describe the structure-activity properties of Leu psi[CH(OH)CH2]Val substitution in other peptidyl ligands of nonviral substrate origin (e.g., angiotensinogen, insulin and pepstatin). Furthermore, the aspartic proteinase selectivities of a few key compounds are summarized relative to evaluation against human renin, human pepsin, and the fungal enzyme, rhizopuspepsin. These studies have led to the rational design of nanomolar potent inhibitors of both HIV-1 and HIV-2 PR. Finally, a 2.5 A resolution X-ray crystallographic structure of U-85548E complexed to synthetic HIV-1 PR dimer (Jaskolski et al., Biochemistry 30, 1600 [1991]) provided a 3-D picture of the inhibitor bound to the enzyme active site, and we performed computer-assisted molecular modeling studies to explore the possible binding modes of the above series of Leu psi[CH(OH)CH2]Val substituted HIV-1 PR inhibitors.

Crystal structure of a complex of HIV-1 protease with a dihydroxyethylene-containing inhibitor: comparisons with molecular modeling.

The structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV-1) protease with a peptide-mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been determined. The inhibitor is Noa-His-Hch psi [CH(OH)CH(OH)]Vam-Ile-Amp (U-75875), and its Ki for inhibition of the HIV-1 protease is < 1.0 nM (Noa = 1-naphthoxyacetyl, Hch = a hydroxy-modified form of cyclohexylalanine, Vam = a hydroxy-modified form of valine, Amp = 2-pyridylmethylamine). The structure of the complex has been refined to a crystallographic R factor of 0.169 at 2.0 A resolution by using restrained least-squares procedures. Root mean square deviations from ideality are 0.02 A and 2.4 degrees, for bond lengths and angles, respectively. The bound inhibitor diastereomer has the R configurations at both of the hydroxyl chiral carbon atoms. One of the diol hydroxyl groups is positioned such that it forms hydrogen bonds with both the active site aspartates, whereas the other interacts with only one of them. Comparison of this X-ray structure with a model-built structure of the inhibitor, published earlier, reveals similar positioning of the backbone atoms and of the side-chain atoms in the P2-P2' region, where the interaction with the protein is strongest. However, the X-ray structure and the model differ considerably in the location of the P3 and P3' end groups, and also in the positioning of the second of the two central hydroxyl groups. Reconstruction of the central portion of the model revealed the source of the hydroxyl discrepancy, which, when corrected, provided a P1-P1' geometry very close to that seen in the X-ray structure.

Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease.

Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment. The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass. Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC. The purified protein retained both RT and RNase H activity, and kinetic parameters (Km and Vmax) were measured with both RNA-dependent DNA polymerization and RNase H activity assays. Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440. Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51.

Chemical synthesis of a biotinylated derivative of the simian immunodeficiency virus protease. Purification by avidin affinity chromatography and autocatalytic activation.

The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.

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.

Zinc inhibition of renin and the protease from human immunodeficiency virus type 1.

We report here for the first time that Zn2+ is an effective inhibitor of renin and the protease from HIV-1, two aspartyl proteinases of considerable physiological importance. Inhibition of renin is noncompetitive and is accompanied by binding of 1 mol of Zn2+/mol of enzyme. Depending on the substrate, inhibition of the HIV protease by Zn2+ can be either competitive or noncompetitive, but in neither case is loss of activity due to disruption of the protease dimer. Inhibition of both enzymes is first order with respect to Zn2+ and is rapidly reversed by addition of EDTA. Ki values are strongly pH dependent and optimal in the range of 20 microM at or above pH 7. All of the data in hand suggest that the inhibitory effect of Zn2+ is a consequence of its binding at, or near, the active-site carboxyl groups of these aspartyl proteinases. This inhibition of the viral enzyme may help to explain some of the beneficial effects seen in AIDS patients who have received Zn2+ therapy.

Actin, troponin C, Alzheimer amyloid precursor protein and pro-interleukin 1 beta as substrates of the protease from human immunodeficiency virus.

We show here for the first time that actin, troponin C, Alzheimer amyloid precursor protein (AAP), and pro-interleukin 1 beta (pro-IL-1 beta), are substrates of the protease encoded by the human immunodeficiency virus (HIV) type-1. As has been seen in other non-viral protein substrates of the HIV protease, the presence of Glu residues in the P2' position appears to play an important role in substrate recognition. Three of the four bonds cleaved in actin, two of the three in troponin C, and all of the bonds hydrolyzed in AAP and pro-IL-1 beta have a P2' Glu residue. In fact, Glu residues are accommodated in all positions from P4 to P4' surrounding the scissile bond in substrates of the HIV proteases, and as many as 4 adjacent Glu residues were seen in one of the bonds cleaved in AAP. This study of non-viral protein substrates has also revealed unexpected amino acids such as Gly, Arg, and Glu in the scissile bond itself rather than the more conventional hydrophobic amino acids. The HIV-2 protease hydrolyzed actin in a manner similar to that of the HIV-1 enzyme, but its cleavage of troponin C was distinct in that it split a bond adjacent to a triplet of Glu residues in P2, P3, and P4 that was refractory to the HIV-1 enzyme. Documentation of cleavage sites in the several important cellular proteins noted above has extended our understanding of the features in a substrate that are recognized by these multi sub-site proteases of retroviral maturation. Moreover, the present work adds to an accumulating body of evidence which demonstrates that these enzymes can damage crucial structural and regulatory cellular proteins if ever their activity is expressed outside the viral particle itself.

The evaluation of non-viral substrates of the HIV protease as leads in the design of inhibitors for AIDS therapy.

Scheme 1 summarizes some of what we have learned from this study of non-viral protein substrates of the HIV proteases. Many of these findings contradict the current understanding of protease specificity. P1-P1' amino acids need not be bulky or hydrophobic and residues at these positions may be even less important than those in flanking positions (e.g., Glu at P2') in dictating the course of hydrolysis. Thus, the pattern of amino acids over the whole binding region must be considered in predicting what will or will not be a substrate of these enzymes and, although we are beginning to understand selectivity at the level of primary structure, a detailed explanation of their specificity is yet to be forthcoming. Nevertheless, studies of this kind find useful application in the design of inhibitors of HIV proteases that will, hopefully, be of value in treatment of AIDS.

Calcium-free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2.

Calcium-free calmodulin-(CaM) is rapidly hydrolyzed by proteases from both human immunodeficiency viruses (HIV) 1 and 2. Kinetic analysis reveals a sequential order of cleavage by both proteases which initiates in regions of the molecule known from X-ray crystallographic analysis of Ca2+/CaM to be associated with calcium binding. Although HIV-1 and HIV-2 proteases hydrolyze two bonds in common, the initial site of cleavage required for subsequent events differs in each case. The first bond hydrolyzed by the HIV-1 protease is the Asn-Tyr linkage in the sequence, -N-I-D-G-D-G-Q-V-N-Y-E-E-, found in the fourth calcium binding loop. In contrast, it is an Ala-Ala bond in the third calcium loop, -D-K-D-G-N-G-Y-I-S-A-A-E-, that is first hydrolyzed by the HIV-2 enzyme, followed in short order by cleavage of the same Asn-Tyr linkage described above. Thereafter, both enzymes proceed to hydrolyze additional peptide bonds, some in common, some not. Considerable evidence exists that inhibitors are bound to the protease in an extended conformation and yet all of the cleavages we observed occur within, or at the beginning of helices in Ca2+/CaM, regions that also appear to be insufficiently exposed for protease binding. Molecular modeling studies indicate that CaM in solution must adopt a conformation in which the first cleavage site observed for each enzyme is unshielded and extended, and that subsequent cleavages involve further unwinding of helices.(ABSTRACT TRUNCATED AT 250 WORDS)