Functionalized N-aryl azetidinones as novel mechanism-based inhibitors of neutrophil elastase

A functionalized N-aryl azetidinone has been shown to inactivate human leukocyte elastase (HLE) and porcine pancreatic elastase (PPE) by an enzyme-mediated process. The inactivation is characterized by the following kinetic constants at pH 8.0 and 37 degrees C: kinact = 0.035 s-1, KI = 1.2 x 10(-4) M for HLE, 0.08 s-1 and 2.7 x 10(-4) M for PPE, respectively. Two parent molecules devoid of the latent leaving group failed to inactivate HLE and PPE and behaved as substrates of these enzymes. A suicide mechanism is postulated involving the formation of an acyl-enzyme and the simultaneous unmasking of a latent quinonimmonium methide ion which irreversibly reacts with an active site nucleophile. Moreover, the inhibitor is still effective at inhibiting elastase preabsorbed onto elastin.     

Irreversible inhibition of serine proteases by peptide derivatives of (alpha-aminoalkyl)phosphonate diphenyl esters

Peptidyl derivatives of diphenyl (alpha-aminoalkyl)phosphonates have been synthesized and are effective and specific inhibitors of serine proteases at low concentrations. Z-PheP(OPh)2 irreversibly reacts with chymotrypsin (kobsd/[I] = 1200 M-1 s-1) and does not react with two elastases. The best inhibitor for most chymotrypsin-like enzymes including bovine chymotrypsin, cathepsin G, and rat mast cell protease II is the tripeptide Suc-Val-Pro-PheP(OPh)2 which corresponds to the sequence of an excellent p-nitroanilide substrate for several chymases. The valine derivative Z-ValP(OPh)2 is specific for elastases and reacts with human leukocyte elastase (HLE, 280 M-1 s-1) but not with chymotrypsin. The tripeptide Boc-Val-Pro-ValP(OPh)2, which has a sequence found in a good trifluoromethyl ketone inhibitor of HLE, is the best inhibitor for HLE (kobsd/[I] = 27,000 M-1 s-1) and porcine pancreatic elastase (PPE, kobsd/[I] = 11,000 M-1 s-1). The rates of inactivation of chymotrypsin by MeO-Suc-Ala-Ala-Pro-PheP(OPh)2 and PPE and HLE by MeO-Suc-Ala-Ala-Pro-ValP(OPh)2 were decreased 2-5-fold in the presence of the corresponding substrate, which demonstrates active site involvement. Only one of two diastereomers of Suc-Val-Pro-PheP(OPh)2 reacts with chymotrypsin (146,000 M-1 s-1), and the enzyme-inhibitor complex had one broad signal at 25.98 ppm in the 31P NMR spectrum corresponding to the Ser-195 phosphonate ester. Phosphonylated serine proteases are extremely stable since the half-time for reactivation was greater than 48 h for the inhibited elastases and 7.5-26 h for chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and in vitro evaluation of pseudosaccharinamine derivatives as potential elastase inhibitors

Pseudosaccharinamine derivatives were evaluated for elastase inhibitory activity. Ester derivatives of pseudosaccharinamine displayed reversible and high inhibition of human leukocyte elastase (HLE) as compared to porcine pancreatic elastase (PPE). Cyanomethyl (2S,3S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylpentanoate was found to inhibit HLE at Ki=0.8 microM.     

Structure of porcine pancreatic elastase complexed with FR901277, a novel macrocyclic inhibitor of elastases, at 1.6 A resolution

Human leukocyte elastase (HLE) is a serine protease that contributes to tissue destruction in various disease states-for example, in emphysema. FR901277 is a natural product isolated from the culture filtrate of Streptomyces resistomicificus and is a potent inhibitor of both HLE and porcine pancreatic elastase (PPE). FR901277 consists of four normal amino acids and three unusual amino acids, and is a unique bicyclic peptide compound. The crystal structure of PPE complexed with FR901277 has been determined at 1.6 A resolution. The Ogamma atom of Ser-195 in PPE did not form a covalent bond with FR901277, but formed a hydrogen bond with the Nvarepsilon atom of His-57. On the other hand, the portion from L-Orn(1) through dehydroxyThr(3) in FR901277 formed an antiparallel beta-sheet structure with the backbone of the active site in PPE. The S4 through S2' binding subsites in PPE were all occupied by the hydrophobic side chains of the inhibitor molecule. Especially, the ethylidene moiety of FR901277 occupied the S1 specific pocket, indicating a CH/pi interaction. In addition, the isopropyl side chain of L-Val(7) was located at the enzyme surface between the S2 and S1' pockets with several van der Waals contacts. However, the amino acid (4) residue was not involved in a significant interaction with PPE. Comparison of inhibitor structures in different environments showed that FR901277 has a highly rigid bicyclic framework; however, it can slightly change its conformation according to the circumstances. The binding mode of FR901277 at the active site of PPE was directly applicable to that in HLE, after consideration of induced fit. The structure of the PPE-FR901277 complex provided much information regarding potential sites for modification of the physicochemical properties of FR901277.     

Crystallization and preliminary diffraction studies of porcine pancreatic elastase in complex with a novel inhibitor

Porcine pancreatic elastase (PPE) was crystallized in complex with a novel inhibitor at pH 5 and X-ray diffraction data were collected at a synchrotron source to 1.66 A. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 50.25 A, b = 57.94 A and c = 74.69 A. PPE is often used as model for drug target, due to its structural homology with the important therapeutic target human leukocyte elastase (HLE). Elastase is a serine protease that belongs to the chymotrypsin family, which has the ability to degrade elastin, an important component in connective tissues. Excessive elastin proteolysis leads to a number of pathological diseases.     

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.     

6-Acylamino-2-1[(ethylsulfonyl)oxy]-1H-isoindole-1,3-diones mechanism-based inhibitors of human leukocyte elastase and cathepsin G: effect of chirality in the 6-acylamino substituent on inhibitory potency and selectivity

Inhibition of human leukocyte elastase(HLE) by a series of 6-acylamino-2-[(ethylsulfonyl)oxy)]-1H-isoindole-1,3-diones was determined and compared to their inhibition of ChT, PPE, and Cat G. The best inhibitor of the series was 6-((1'S)-camphanyl)amino-2-[(ethylsulfonyl) oxy]-1H-isoindole-1,3-dione 5b, with a k(obs)/[I] = 11,000 M(-1) s(-1). This study revealed that HLE shows a preference for the S stereochemistry and tolerates hydrophobic substituents in the Sn' binding sites. Molecular modeling of non-covalent HLE-inhibitor complexes was used as a tool to investigate our binding model. Buffer stability assays reveal that these compounds are susceptible to hydrolysis at physiological pH.     

Reversible, slow, tight-binding inhibition of human leukocyte elastase

CBz-Ala-Ala-Pro-ambo-Val-CF3 (1) was synthesized. The compound inhibits human Leucocyte elastase with Ki = 1.0 x 10(-9) M. This inhibitor is reversible, slow, tight-binding inhibitor with k on = 2 x 10(4) M-1 s-1 k off = 1.9 x 10(-5) s-1. For the solubilization of elastin by HLE by 1 I.C. 50 = 110 nM. This inhibitor is the most effective aldehyde or ketone inhibitor of a serine proteinase yet described.     

Elastinolytic activity of horse leukocyte proteinases. Comparison with elastases from human leukocytes and porcine pancreas

Three proteinases from the azurophilic granules of horse leucocytes are typical elastases degrading elastin at neutral pH. Both proteinases: 1 and 2A exhibit similar elastinolytic activity, comparable with human leucocyte elastase (HLE). In relation to human enzyme, elastase 2B shows several-fold higher activity, which is comparable to the porcine pancreatic elastase activity (PPE). Similarly to HLE elastinolytic activity of the horse proteinases increases at higher ionic strength: twofold in case of 1 or 2A and fivefold for 2B. Significant activity observed during degradation of homologous lung elastin, implies the possible role of these enzymes during pathological injury of connective tissue in the lower respiratory tract and suggests similar pathogenesis of horse and human pulmonary emphysema.     

Susceptibility of baboon aorta elastin to proteolysis

Elastin was purified from baboon aorta using Achromobacter collagenase and its susceptibility to proteolysis by various enzymes was studied. Human leukocyte elastase (HLE) hydrolysed baboon aortic elastin 8 times faster than human cathepsin G. Bovine chymotrypsin had virtually no activity against this substrate. The kinetic constants V and [S50] of aortic elastin hydrolysis by HLE (0.15 microM) were 0.00286 mg x ml-1 x min-1 and 0.158 mg x ml-1, respectively. One mg of this elastin could be saturated with 5.6 micrograms of HLE. As with elastins isolated from other sources, the hydrolysis of baboon aortic elastin by HLE was highly sensitive to ionic strength, and a biphasic effect was obtained with increasing NaCl concentrations. A nearly 2-fold stimulation of elastolysis was observed at a 0.15M NaCl concentration. Further increase in ionic strength led to a continuous decrease of the rate of elastolysis which paralleled the decrease of adsorption of elastase to baboon aortic elastin. Cathepsin G, but not bovine alpha-chymotrypsin, was able to stimulate the rate of hydrolysis of baboon aortic elastin by HLE. A 1.7 fold stimulation was observed for a 1:1 molar ratio of the two proteinases and rose to 2.1 for a HLE/Cat. G ratio equal to 8.     

Structure-activity relationships for the selectivity of hepatitis C virus NS3 protease inhibitors

The selectivity of hepatitis C virus (HCV) non-structural protein 3 (NS3) protease inhibitors was determined by evaluating their inhibitory effect on other serine proteases (human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine pancreatic chymotrypsin (BPC)) and a cysteine protease (cathepsin B). For these peptide inhibitors, the P1-side chain and the C-terminal group were the major determinants of selectivity. Inhibitors with electrophilic C-terminal residues were generally non-selective while compounds with non-electrophilic C-terminal residues were more selective. Furthermore, compounds with P1 aminobutyric acid residues were non-selective, while 1-aminocyclopropane-1-carboxylic acid (ACPC) and norvaline-based inhibitors were generally selective. The most potent and selective inhibitors of NS3 protease tested contained a non-electrophilic phenyl acyl sulfonamide C-terminal residue. HLE was most likely to be inhibited by the HCV protease inhibitors, in agreement with similar substrate specificities for these enzymes. The identified structure-activity relationships for selectivity are of significance for design of selective HCV NS3 protease inhibitors.     

Structure-activity studies of fluoroketone inhibitors of alpha-lytic protease and human leukocyte elastase

We have synthesized a series of peptidyl fluoroketones that reversibly inhibit the serine proteases human leukocyte elastase (HLE) and alpha-lytic protease (alpha-LP). Ac-ambo-AlaCF3 (1) inhibits HLE and alpha-LP with Ki's of 2.4 and 15 mM, respectively. The effects of structural variations on this parent compound on Ki and the kinetics of inhibition were studied. The acetyl group was replaced by the tripeptide Z-L-Ala-L-Ala-L-Pro to yield the tetrapeptide trifluoroketone (TFK) Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF3 (2). This extension reduced Ki 3500-fold for HLE and 3000-fold for alpha-LP. Removal of a fluorine atom from a TFK decreases Ki about 15- to 30-fold with both enzymes. Replacement of one fluorine atom of 2 by a residue (-CH2-CH2-COLeuOMe) (6) which can interact with the S'1 and S'2 subsites decreased Ki 30-fold for HLE and 150-fold for alpha-LP compared to Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF2H (3). The Ki of 6 for HLE is approximately equal to that of trifluoroketone 2. For alpha-LP Ki of 6 is 10-fold lower than that for the trifluoroketone 2. Inhibitors with Ki values less than 10(-7) M exhibit slow binding kinetics. By analogy to cholinesterases and chymotrypsin, it is likely that these enzymes combine with the keto form of the inhibitor to form the enzyme-inhibitor complex. Therefore, kon and Ki were corrected for the ketone concentration. The corrected kon values for the slow binding inhibitors are in most cases less than diffusion controlled, ranging between 8.2 X 10(4) and 4.68 X 10(6) M-1 s-1. An exception is Z-L-Ala-L-Ala-L-Pro-ambo-ValCF3 (8) where kon = 9 X 10(7) M-1 s-1, which is nearly diffusion controlled.     

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.     

Proline-valine pseudo peptide enol lactones. Effective and selective inhibitors of chymotrypsin and human leukocyte elastase

Pro-Val pseudo dipeptides incorporating protio and halo enol lactones were tested for inhibitory activity against the serine proteases human leukocyte elastase (HLE), porcine pancreatic elastase, alpha-chymotrypsin, trypsin, thrombin, and urokinase. The protio enol lactones 1a-c were found to be HLE substrates but were poor alternate substrate inhibitors. The bromo enol lactone trans isomer 2a was found to be a very effective inhibitor of HLE and chymotrypsin, as shown by the binding constants (KI), acylation rates (ka), inactivation rates, and partition ratios determined for each enzyme. This inhibitor shows better specificity toward its target enzyme HLE than monosubstituted halo enol lactones; we attribute this to a pseudo dipeptide acyl enzyme whose structure is similar to that adopted by good peptide substrates of HLE. Inactivation of chymotrypsin by the bromo enol lactone 2a is permanent, but inactivation of HLE is partially recoverable upon treatment with the nucleophile hydrazine, indicating that lactone 2a produces two species of inactivated HLE. The more stable of these species could be the result of alkylation of His-57 by the electrophilic bromomethyl ketone revealed in the acyl enzyme, and the less stable, hydrazine-reactivatable species could be the result of alkylation of Asp-102 or the hydrolysis of the bromomethyl ketone group in the initially formed acyl enzyme to form a new, more stable acyl enzyme.     

An elastase-dependent pathway of plasminogen activation

In reaction mixtures containing Glu-plasminogen, alpha 2-antiplasmin, and tissue plasminogen activator or urokinase, either pancreatic or leukocyte elastase enhances the rate of plasminogen activation by 2 or more orders of magnitude. This effect is the consequence of several reactions. (a) In concentrations on the order of 100 nM, elastase degrades plasminogen within 10 min to yield des-kringle1-4-plasminogen (mini-plasminogen), which is 10-fold more efficient than Glu-plasminogen as a substrate for plasminogen activators. Des-kringle1-4-plasminogen is insensitive to cofactor activities of fibrin(ogen) fragments or an endothelial cell cofactor. (b) Des-kringle1-4-plasmin is one-tenth as sensitive as plasmin to inhibition by alpha 2-antiplasmin: k" = 10(6) M-1 s-1 versus 10(7) M-1 s-1. (c) alpha 2-Antiplasmin is disabled efficiently by elastase, with a k" of 20,000 M-1 s-1. The elastase-dependent reactions are not influenced by 6-aminohexanoate. In diluted (10-fold) blood plasma, the capacity of endogenous inhibitors to block plasmin expression is suppressed by 30 microM elastase. It is proposed that elastases provide an alternative pathway for Glu-plasminogen activation and a mechanism for controlling initiation of fibrinolysis by urokinase-type plasminogen activators.     

Distinct binding sites of Ala48-hirudin1-47 and Ala48-hirudin48-65 on alpha-thrombin

The interaction of alpha-thrombin with Ala48-hirudin, Ala48-hirudin1-47, and Ala48-hirudin48-65 was analyzed. Mutations at Pro48 were found to cause only slight changes in the kon (human: 3.1 +/- 0.3 x 10(8) M-1 s-1; bovine: 1.03 +/- 0.3 x 10(8) M-1 s-1) and koff (human: 0.4 +/- 0.2 x 10(-3) s-1; bovine: 2.9 +/- 0.4 x 10(-3) s-1) rate constants for the formation of the thrombin-hirudin complex. The amino-terminal fragment Ala48-hirudin1-47 containing the three disulfide bridges and the carboxyl-terminal fragment Ala48-hirudin48-65 were derived from the Ala48 mutant by proteolysis with endoproteinase Lys-C. These fragments inhibit bovine alpha-thrombin clotting activity with IC50 values of 0.6 and 4.9 microM, respectively (2.4 nM for r-hirudin). By mapping the interaction of Ala48-hirudin-derived fragments with bovine alpha-thrombin by limited proteolysis with trypsin and pancreatic elastase distinct binding sites for each fragment were determined. The carboxyl-terminal fragment was found to bind to the proposed anion-binding exosite in the region B62-74, whereas the amino-terminal fragment binds to a region around the elastase cleavage site at residues 150-151 of the alpha-thrombin B-chain.     

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

The crystal structure of the complex of non-peptidic inhibitor of human neutrophil elastase ONO-6818 and porcine pancreatic elastase

The crystal structure of a new inhibitor of human neutrophil elastase (HNE), N-[2-[5-(tert-butyl)-1,3,4-oxadiazol-2-yl]-(IRS)-1-(methylethyl)-2-oxoethyl]-2-(5-amino-6-oxo-2-phenyl-6H-pyrimidin-1-ly)acetamide (ONO-6818, 1) complexed to porcine pancreatic elastase (PPE) has been determined at 1.86 A resolution. Analytical results provided evidence of a 1:1 complex in which the electrophilic ketone of 1 covalently bound to O gamma of Ser195 at the active site of PPE. The role of the unique electron-withdrawing ketone of 1 has been elucidated.     

Kinetics of the inhibition of free and elastin-bound human pancreatic elastase by alpha 1-proteinase inhibitor and alpha 2-macroglobulin

At pH 8.0 and 25 degrees C alpha 1-proteinase inhibitor and alpha 2-macroglobulin bind human pancreatic elastase with rate constants of 4.7.10(5) M-1.s-1 and 6.4.10(6) M-1.s-1, respectively. The corresponding delay times of elastase inhibition in plasma are 0.4 s and 0.2 s, respectively, indicating that both inhibitors may act as physiological antielastases. Elastin impairs the elastase inhibitory capacity of alpha 1-proteinase inhibitor and alpha 2-macroglobulin. In presence of human elastin, the former behaves like a slow-binding elastase inhibitor, with a rate constant of about 260 M-1.s-1. In contrast, alpha 2-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin.     

The α-test: rapid cell-free CD4 enumeration using whole saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides. Thus, identification of cell-free correlates that directly regulate the number of CD4(+) T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates. The number of stem cells that enter blood and are destined to become circulating CD4(+) T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLE(CS)) and the HLE(CS)-reactive active α(1)proteinase inhibitor (α(1)PI, α(1)antitrypsin, SerpinA1). In HIV-1 disease, α(1)PI is inactivated due to disease processes. In the early asymptomatic categories of HIV-1 disease, active α(1)PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 μM, and to achieve normal levels during the symptomatic categories. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with >220 CD4 cells/μl, CD4 counts were correlated with serum levels of active α(1)PI (r(2)=0.93, p<0.0001, n=26) and inactive α(1)PI (r(2)=0.91, p<0.0001, n=26). Administration of α(1)PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting α(1)PI participates in regulating the number of CD4(+) T cells in blood. With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active α(1)PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum α(1)PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to α(1)PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA(3)NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active α(1)PI in saliva. The resulting inhibition of PPE by active α(1)PI can be measured by adding the PPE substrate SA(3)NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/μl), the concentration of α(1)PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable. Thus, active α(1)PI in saliva is calculated as a ratio to saliva protein content and is termed the α(1)PI Index. Results presented herein demonstrate that the α(1)PI Index provides an accurate and precise physiologic method for calculating CD4 counts.