HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3 subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition. rational drug design ͉ aspartic proteases ͉ carboranes ͉ x-ray structure analysis ͉ virostatics

Propeptide blocks the active site in the inactive zymogen of cathepsin D and is cleaved off during zymogen activation. We have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin D using a kinetic assay. Our mapping of the cathepsin D propeptide indicated two domains in the propeptide involved in the inhibitory interaction with the enzyme core: the active site "anchor" domain and the N-terminus of the propeptide. The latter plays a dominant role in propeptide inhibition (nanomolar Ki), and its high-affinity binding was corroborated by fluorescence polarization measurements. In addition to the inhibitory domains of propeptide, a fragment derived from the N-terminus of mature cathepsin D displayed inhibition. This finding supports its proposed regulatory function. The interaction mechanisms of the identified inhibitory domains were characterized by determining their modes of inhibition as well as by spatial modeling of the propeptide in the zymogen molecule. The inhibitory interaction of the N-terminal propeptide domain was abolished in the presence of sulfated polysaccharides, which interact with basic propeptide residues. The inhibitory potency of the active site anchor domain was affected by the Ala38pVal substitution, a propeptide polymorphism reported to be associated with the pathology of Alzheimer's disease. We infer that propeptide is a sensitive tethered ligand that allows for complex modulation of cathepsin D zymogen activation.

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Activity-based proteomics employing active-site-directed chemical probes for enzymatic activity profiling in complex proteomes has greatly accelerated the functional annotation of proteins. [1,2] In protease research, new generations of activitybased probes have led to tremendous progress in our understanding of the biochemistry and physiology of cysteine peptidases over the last decade. [3,4] These probes were designed for in vitro applications as well as for in vivo monitoring in living cells and whole animals, and has directed research towards biomarker discovery and drug screening. However, the current availability of analogous chemical tools selective for other classes of peptidases is limited. The situation is especially critical in the case of aspartic peptidases, despite the fact that these enzymes play an important role in a number of devastating human pathologies, such as AIDS, Alzheimer's disease, and cancer. A major reason for the limited number of available probes for aspartic peptidases is the lack of suitable covalent inhibitors that can be used as starting templates for probe derivation. There are only a few examples of recent advances in this field, including probes for human g-secretase and malaria plasmepsins that were constructed based on the concept of photoaffinity labeling. [5,6] In this work, we focus on cathepsin D, an aspartic peptidase that is critically associated with cancer development and progression through its complex proteolytic and proliferative action and is a prognostic marker in breast cancer (for a recent review, see ref. [7]). There is still insufficient knowledge regarding the interaction partners and activity regulation mechanisms of this therapeutic target, and the introduction of novel imaging tools is required for these studies.Here, we present a convenient synthesis of novel proteomicactivity-based probes for cathepsin D that are suitable for applications in activity profiling and analysis of post-translational activation processing.The design of three probes, sBAP-09, dBAP-09, and FAP-09, is shown in Figure 1. Their structures consist of three functionalities: 1) a peptidic binding core, 2) a photoreactive group as a probe "warhead", and 3) a fluorescent group or biotin as a reporter tag. The binding core was constructed by using pepstatin, a reversible peptidomimetic inhibitor of aspartic peptidases, as a template. The pepstatin molecule was structurally minimized and optimized for cathepsin D inhibition [8] to yield the binding core sequence shown in Figure 1. The statin residue in the peptidic core interacts with the enzyme's catalytic residues and acts as a transition-state mimic. The binding core scaffold was modified at its N and/or C terminus to incorporate functionalities 2) and 3). The crystal structure of human cathepsin D in complex with pepstatin (PDB ID: 1LYB) suggests that bulky substituents in the terminal positions will protrude from the enzyme active site and should not interfere with the interaction of ligand and enzyme. In the sBAP-09 and dBAP-09 p...