Protease Variants Research Articles

An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding

The extreme thermal stabilization achieved by the introduction of a disulfide bond (G8C/N60C) into the cysteine-free wild-type-like mutant (pWT) of the neutral protease from Bacillus stearothermophilus[Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, Van den Burg B, Venema G, Ulbrich-Hofmann R & Eijsink VG (1997) J Biol Chem272, 11152-11156] was attributed to the fixation of the loop region 56-69. In this study, the role of calcium ions in the guanidine hydrochloride (GdnHCl)-induced unfolding and autoproteolysis kinetics of pWT and G8C/N60C was analyzed by fluorescence spectroscopy, far-UV CD spectroscopy and SDS/PAGE. First-order rate constants (kobs) were evaluated by chevron plots (ln kobs vs. GdnHCl concentration). The kobs of unfolding showed a difference of nearly six orders of magnitude (DeltaDeltaG# = 33.5 kJ.mol(-1) at 25 degrees C) between calcium saturation (at 100 mM CaCl2) and complete removal of calcium ions (in the presence of 100 mM EDTA). Analysis of the protease variant W55F indicated that calcium binding-site III, situated in the critical region 56-69, determines the stability at calcium ion concentrations between 5 and 50 mM. In the chevron plots the disulfide bridge in G8C/N60C shows a similar effect compared with pWT as the addition of calcium ions, suggesting that the introduced disulfide bridge fixes the region (near calcium binding-site III) that is responsible for unfolding and subsequent autoproteolysis. Owing to the presence of the disulfide bridge, the DeltaDeltaG# is 13.2 kJ.mol(-1) at 25 degrees C and 5 mM CaCl2. Non-linear chevron plots reveal an intermediate in unfolding probably caused by local unfolding of the loop 56-69. The occurrence of this intermediate is prevented by calcium concentrations of > 5 mM, or the introduction of the disulfide bridge G8C/N60C.

Molecular models of NS3 protease variants of the Hepatitis C virus

BackgroundHepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.ResultsThe atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures.ConclusionsThis project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.

A particular hepatitis C virus protease NS3 gene pattern in a patient not responding to interferon-ribavirin therapy

Hepatitis C virus (HCV) is characterized by a high degree of genetic heterogeneity. This is a consequence of its rapid turnover in vivo and of the low fidelity of its RNAdependent RNA polymerase, which lacks proofreading activity. Its high propensity to mutate is also reflected by the existence of closely related genomes, known as quasispecies [1]. NS3 protease is essential for HCV replication. It acts via a catalytic triad consisting of a histidine, an aspartate and a serine. It is also one of the most promising targets for specific anti-HCV therapy [2]. Although the NS3 protease is considered to be one of the least variable genes in the HCV genome, we previously found an appreciable polymorphism in this genomic region in 294 HCV genotype 1 clones isolated from the serum of 17 infected patients [3]. Reported here is a particular pattern, including three deletions and one insertion, found in four clones among 19 NS3 protease variants of a quasispecies detected in one of these patients.

Structural and thermodynamic basis for the binding of TMC114, a next-generation human immunodeficiency virus type 1 protease inhibitor.

TMC114, a newly designed human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is extremely potent against both wild-type (wt) and multidrug-resistant (MDR) viruses in vitro as well as in vivo. Although chemically similar to amprenavir (APV), the potency of TMC114 is substantially greater. To examine the basis for this potency, we solved crystal structures of TMC114 complexed with wt HIV-1 protease and TMC114 and APV complexed with an MDR (L63P, V82T, and I84V) protease variant. In addition, we determined the corresponding binding thermodynamics by isothermal titration calorimetry. TMC114 binds approximately 2 orders of magnitude more tightly to the wt enzyme (K(d) = 4.5 x 10(-12) M) than APV (K(d) = 3.9 x 10(-10) M). Our X-ray data (resolution ranging from 2.2 to 1.2 A) reveal strong interactions between the bis-tetrahydrofuranyl urethane moiety of TMC114 and main-chain atoms of D29 and D30. These interactions appear largely responsible for TMC114's very favorable binding enthalpy to the wt protease (-12.1 kcal/mol). However, TMC114 binding to the MDR HIV-1 protease is reduced by a factor of 13.3, whereas the APV binding constant is reduced only by a factor of 5.1. However, even with the reduction in binding affinity to the MDR HIV protease, TMC114 still binds with an affinity that is more than 1.5 orders of magnitude tighter than the first-generation inhibitors. Both APV and TMC114 fit predominantly within the substrate envelope, a property that may be associated with decreased susceptibility to drug-resistant mutations relative to that of first-generation inhibitors. Overall, TMC114's potency against MDR viruses is likely a combination of its extremely high affinity and close fit within the substrate envelope.

Determining and overcoming resistance to HIV protease inhibitors.

HIV protease represents a major target for development of antiviral therapeutics. The introduction of HIV protease (PR) inhibitors (PIs) to clinical practice and the application of highly active antiretroviral therapy resulted in decreased mortality and prolonged life expectancy of HIV-positive patients. However, the high polymorphism of HIV leads to rapid selection of viral variants resistant towards the inhibitors. Such resistant PR variants have developed in HIV-positive patients after treatment with any of the eight PIs approved for clinical use. In this review we overview (i) the methods for the identification and assessment of viral resistance in HIV positive patients, and (ii) the approaches medicinal chemists take to overcome it. Rational antiviral therapy brings about the need for quantitative assessment of the level of drug resistance development in the course of the treatment. At present, two main approaches are taken: in genotypic assays the viral sequences are PCR amplified, sequenced and changes in the viral gene sequence known to be associated with reduced drug sensitivity are identified, while phenotypic assays test the ability of a virus to grow in the presence of a drug or combination of drugs. The advantages and drawbacks of these methods, as well as their relevance for the therapy are discussed. We also review the efforts to design second-generation PIs, capable of potently inhibiting multi-resistant HIV-1 PR species, using structure-assisted design of the compounds targeted to the active site, as well as alternative approaches with compounds binding to other domains of the PR molecule.

Characterisation of Mutated Proteinases Derived from HIV-Positive Patients: Enzyme Activity, Vitality and Inhibition

HIV protease (PR) specifically cleaves viral polyproteins to yield infectious progeny virus particles. Inactivation of PR leads to loss of virus infectivity and PR thus became an attractive pharmaceutic target. Indeed, seven protease inhibitors (PI) have been approved for clinical use to date. However, emerging resistant viral variants with reduced sensitivity to PIs become a major obstacle to successful control of viral replication. We have previously reported the design, testing and structural analysis of a pseudopeptide inhibitor, QF34, which efficiently inhibits a wide variety of PR variants. In a clinical study, we have monitored more than 100 HIV-positive patients in the Czech Republic undergoing highly active antiretroviral therapy including PI. In this paper we describe kinetic characterisation of two highly resistant PR species isolated from these patients. The mutated proteases accumulated as much as 14 amino acid exchanges and develop resistance to saquinavir, ritonavir, indinavir and nelfinavir with vitality value up to 150. Kinetic analyses revealed that second-generation PI lopinavir and QF34 retained their subnanomolar potency against both multidrug resistant PR variants. These results suggest a route to the design of PIs capable of inhibiting a variety of resistant PR mutants.

Human immunodeficiency virus (HIV) type 1 transframe protein can restore activity to a dimerization-deficient HIV protease variant.

The protease (PR) from human immunodeficiency virus (HIV) is essential for viral replication: this aspartyl protease, active only as a dimer, is responsible for cleavage of the viral polyprotein precursors (Gag and Gag-Pol), to release the functional mature proteins. In this work, we have studied the structure-function relationships of the HIV PR by combining a genetic test to detect proteolytic activity in Escherichia coli and a bacterial two-hybrid assay to analyze PR dimerization. We showed that a drug-resistant PR variant isolated from a patient receiving highly active antiretroviral therapy is impaired in its dimerization capability and, as a consequence, is proteolytically inactive. We further showed that the polypeptide regions adjacent to the PR coding sequence in the Gag-Pol polyprotein precursor, and in particular, the transframe polypeptide (TF), located at the N terminus of PR, can facilitate the dimerization of this variant PR and restore its enzymatic activity. We propose that the TF protein could help to compensate for folding and/or dimerization defects in PR arising from certain mutations within the PR coding sequence and might therefore function to buffer genetic variations in PR.

In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor.

The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

Processivity and drug-dependence of HIV-1 protease

To investigate the role of processivity and drug-dependence of HIV-1 protease as fitness determinants in variants resistant to protease inhibitors (PI).HIV-1 protease sequences from 32 infected subjects (27 patients who failed PI-treatments and five PI-naive controls) were evaluated using a recombinant method. The HIV-1 phenotype to seven PI was analysed together with the replication capacity of recombinants and the processivity and drug-dependence of the HIV-1 proteases. Protease mutants (positions 10, 46, 54, 82, 84, 90, and combinations thereof) were generated in vitro and studied under identical experimental conditions.In the absence of PI, 24 of 27 (89%) resistant proteases from treated subjects showed decreased processivity compared with the wild type. Processivity was lower in sequences bearing fewer mutations, than in more mutated ones. Twelve sequences (44%) conferred slower replication kinetics to the recombinant viruses. Seven sequences (26%) showed higher processivity levels in the presence of PI than in their absence, suggesting that drug-dependence influences PI-resistant variants. Among the mutants generated in vitro, mutations 82A and 90M determined broad cross-resistance to PI in association with 10I. A drop of processivity was observed for the 82A+90M variants; 10I allowed partial recovery for 82A and 84V, and marked recovery for 90M mutants.A decrease in HIV-1 protease processivity parallels early selection of primary mutations, whereas its recovery is driven by compensatory mutations. Furthermore, a PI may select drug-dependent, besides resistant, HIV-1 protease variants. Changes in processivity and drug-dependence of HIV-1 proteases have implications in the replication capacity of PI-resistant viruses.

HIV-1 protease variants from 100-fold drug resistant clinical isolates: expression, purification, and crystallization

High-resolution X-ray crystallographic structures of HIV-1 protease clinical variants complexed with licensed inhibitors are essential to understanding the fundamental cause of protease drug resistance. There is a need for structures of naturally evolved HIV-1 proteases from patients failing antiretroviral therapy. Here, we report the expression, purification, and crystallization of clinical isolates of HIV-1 protease that have been characterized to be more than 100 times less susceptible to US FDA approved protease inhibitors.

Elucidation of HIV-1 protease resistance by characterization of interaction kinetics between inhibitors and enzyme variants

The kinetics of the interaction between drug-resistant variants of HIV-1 protease (G48V, V82A, L90M, I84V/L90M, and G48V/V82A/I84V/L90M) and clinically used inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) were determined using biosensor technology. The enzyme variants were immobilized on a biosensor chip and the association and dissociation rate constants ( k on and k off) and affinities ( K D) for interactions with inhibitors were determined. A unique interaction kinetic profile was observed for each variant/inhibitor combination. Substitution of single amino acids in the protease primarily resulted in reduced affinity through increased k off for the inhibitors. For inhibitors characterized by fast association rates to wild-type protease (ritonavir, amprenavir, and indinavir), additional substitutions resulted in a further reduction of affinity by a combination of decreased k on and increased k off. For inhibitors characterized by slow dissociation rates to wild-type enzyme (saquinavir and nelfinavir), the decrease of affinity conferred by additional mutations was attributed to increased k off values. Development of resistance thus appears to be associated with a change of the distinctive kinetic parameter contributing to high affinity. Further inhibitor design should focus on improving the “weak point” of the lead compound, that being either k on or k off.

HIV-1 Vif-Derived Peptide Inhibits Drug-Resistant HIV Proteases

Vif, one of the six accessory genes expressed by HIV-1, is essential for the productive infection of natural target cells. Previously we suggested that Vif acts as a regulator of the viral protease (PR): It prevents the autoprocessing of Gag and Gag-Pol precursors until virus assembly, and it may control the PR activity in the preintegration complex at the early stage of infection. It was demonstrated before that Vif, and specifically the 98 amino acid stretch residing at the N′-terminal part of Vif (N′-Vif), inhibits both the autoprocessing of truncated Gag-Pol polyproteins in bacterial cells and the hydrolysis of synthetic peptides by PR in cell-free systems. Linear synthetic peptides derived from N′-Vif specifically inhibit and bind HIV-1 PR in vitro, and arrest virus production in tissue culture. Peptide mapping of N′-Vif revealed that Vif88-98 is the most potent PR inhibitor. Here we report that this peptide inhibits both HIV-1 and HIV-2, but not ASLV proteases in vitro. Vif88-98 retains its inhibitory effect against drug-resistant HIV-1 PR variants, isolated from patients undergoing long-term treatment with anti-PR drugs. Variants of HIV protease bearing the mutation G48V are resistant to inhibition by this Vif-derived peptide, as shown by in vitro assays. In agreement with the in vitro experiments, Vif88-98 has no effect on the production of infectious particles in cells infected with a G48V mutated virus.

Lack of synergy for inhibitors targeting a multi-drug-resistant HIV-1 protease.

The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to approximately 2.2 A resolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.

Altered substrate specificity of drug-resistant human immunodeficiency virus type 1 protease.

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV.

In Vitro Characterization of a Purified NS2/3 Protease Variant of Hepatitis C Virus

The cleavage of the hepatitis C virus polyprotein between the nonstructural proteins NS2 and NS3 is mediated by the NS2/3 protease, whereas the NS3 protease is responsible for the cleavage of the downstream proteins. Purification and in vitro characterization of the NS2/3 protease has been hampered by its hydrophobic nature. NS2/3 protease activity could only be detected in cells or in in vitro translation assays with the addition of microsomal membranes or detergent. To facilitate purification of this poorly characterized protease, we truncated the N-terminal hydrophobic domain, resulting in an active enzyme with improved biophysical properties. We define a minimal catalytic region of NS2/3 protease retaining autocleavage activity that spans residues 904-1206 and includes the C-terminal half of NS2 and the N-terminal NS3 protease domain. The NS2/3 (904-1206) variant was purified from Escherichia coli inclusion bodies and refolded by gel filtration chromatography. The purified inactive form of NS2/3 (904-1206) was activated by the addition of glycerol and detergent to induce autocleavage at the predicted site between Leu(1026) and Ala(1027). NS2/3 (904-1206) activity was dependent on zinc ions and could be inhibited by NS4A peptides, peptides that span the cleavage site, or an N-terminal peptidic cleavage product. This NS2/3 variant will facilitate the development of an assay suitable for identifying inhibitors of HCV replication.

Viral evolution in response to the broad-based retroviral protease inhibitor TL-3.

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

Engineering a Hyperstable Enzyme by Manipulation of Early Steps in the Unfolding Process

Protein engineering experiments have recently yielded hyperstable variants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). These variants contain mutations suggested by comparison of TLP-ste with its more thermostable counterpart thermolysin, as well as rationally designed mutations. The key to the successful stabilization strategy was the identification of a “weak” region that is involved in early unfolding events (“unfolding region”). Mutations in this region had large effects on stability, whereas mutations in other parts of the protein generally had minor effects. The mutational strategies that were used as well as characteristics of the engineered hyperstable biocatalysts are reviewed below.

Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli.

We describe a genetic system that allows in vivo screening or selection of site-specific proteases and of their cognate-specific inhibitors in Escherichia coli. This genetic test is based on the specific proteolysis of a signaling enzyme, the adenylate cyclase (AC) of Bordetella pertussis. As a model system we used the human immunodeficiency virus (HIV) protease. When an HIV protease processing site, p5, was inserted in frame into the AC polypeptide, the resulting ACp5 protein retained enzymatic activity and, when expressed in an E. coli cya strain, restored the Cya(+) phenotype. The HIV protease coexpressed in the same cells resulted in cleavage and inactivation of ACp5; the cells became Cya(-). When the entire HIV protease, including its adjacent processing sites, was inserted into the AC polypeptide, the resulting AC-HIV-Pr fusion protein, expressed in E. coli cya, was autoproteolysed and inactivated: the cells displayed Cya(-) phenotype. In the presence of the protease inhibitor indinavir or saquinavir, AC-HIV-Pr autoproteolysis was inhibited and the AC activity of the fusion protein was preserved; the cells were Cya(+). Protease variants resistant to particular inhibitors could be easily distinguished from the wild type, as the cells displayed a Cya(-) phenotype in the presence of these inhibitors. This genetic test could represent a powerful approach to screen for new proteolytic activities and for novel protease inhibitors. It could also be used to detect in patients undergoing highly active antiretroviral therapy the emergence of HIV variants harboring antiprotease-resistant proteases.

Structural and biochemical features distinguish the foot-and-mouth disease virus leader proteinase from other papain-like enzymes

The structures of the two leader protease (Lpro) variants of foot-and-mouth disease virus known to date were solved using crystals in which molecules were organized as molecular fibers. Such crystals diffract to a resolution of only approximately 3 Å. This singular, pseudo-polymeric organization is present in a new Lpro crystal form showing a cubic packing. As molecular fiber formation appeared unrelated to crystallization conditions, we mutated the reactive cysteine 133 residue, which makes a disulfide bridge between adjacent monomers in the fibers, to serine. None of the intermolecular contacts found in the molecular fibers was present in crystals of this variant. Analysis of this Lpro structure, refined at 1.9 Å resolution, enables a detailed definition of the active center of the enzyme, including the solvent organization. Assay of Lpro activity on a fluorescent hexapeptide substrate showed that Lpro, in contrast to papain, was highly sensitive to increases in the cation concentration and was active only across a narrow pH range. Examination of the Lpro structure revealed that three aspartate residues near the active site, not present in papain-like enzymes, are probably responsible for these properties.

Inhibition of HIV-1 protease by a boron-modified polypeptide

Six boronated tetrapeptides with the carboxy moiety of phenylalanine replaced by dihydroxyboron were synthesized, and their activities against human immunodeficiency virus 1 (HIV-1) protease subsequently investigated. The sequences of these peptides were derived from HIV-1 protease substrates, which included the C-terminal part of the scissile bond (Phe–Pro) within the gag–pol polyprotein. Enzymatic studies showed that these compounds were competitive inhibitors of HIV-1 protease with K i values ranging from 5 to 18 μM when experiments were performed at high enzyme concentrations (above 5 × 10 −8 M); however, at low protease concentrations inhibition was due in part to an increase of the association constants of the protease subunits. Ac-Thr-Leu-Asn-PheB inhibited HIV-1 protease with a K i of 5 μM, whereas the non-boronated parental compound was inactive at concentrations up to 400 μM, which indicates the significance of boronation in enzyme inhibition. The boronated tetrapeptides were inhibitory to an HIV-1 protease variant that is resistant to several HIV-1 protease inhibitors. Finally, fluorescence analysis showed that the interactions between the boronated peptide Ac-Thr-Leu-Asn-PheB and HIV-1 protease resulted in a rapid decrease of fluorescence emission at 360 nm, which suggests the formation of a compound/enzyme complex. Boronated peptides may provide useful reagents for studying protease biochemistry and yield valuable information toward the development of protease dimerization inhibitors.