Complex modulation of peptidolytic activity of cathepsin D by sphingolipids.

Cathepsin D is an aspartic peptidase involved in cellular processes including proliferation and apoptosis and implicated in human pathologies such as cancer and neurodegeneration. Our knowledge about the relationship between proteolysis and bioactive sphingolipids is still very limited. Here, we describe a complex pattern of modulation of the peptidolytic activity of cathepsin D by sphingolipids. A panel of sphingolipid derivatives was screened in a FRET-based assay; these molecules demonstrated negative or positive modulation of cathepsin D peptidolytic activity, depending on the sphingolipid structure. Certain sphingosines and ceramides inhibited cathepsin D in the submicromolar range, and structural requirements for this inhibitory effect were evaluated. The interaction of cathepsin D with sphingolipids was also demonstrated by fluorescence polarization measurements and determined to follow a competitive inhibition mode. In contrast, monoester phosphosphingolipids, especially ceramide-1-phosphate, were identified as activators of cathepsin D peptidolytic activity at submicromolar concentrations. Thus, sphingolipids and phosphosphingolipids, known to be antagonistic in their cell-signaling functions, displayed opposite modulation of cathepsin D. Sphingolipid-based modulators of cathepsin D are potentially involved in the control of cathepsin D-dependent processes and might serve as a scaffold for the development of novel regulators of this therapeutic target.

Cathepsin D propeptide: mechanism and regulation of its interaction with the catalytic core.

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.

From nonpeptide toward noncarbon protease inhibitors: metallacarboranes as specific and potent inhibitors of HIV protease.

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 K(i) value of 2.2 nM and a submicromolar EC(50) 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 A 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.

Activation processing of cathepsin H impairs recognition by its propeptide.

Free propeptides are known to function as inhibitors of the parental mature cysteine cathepsins. This general rule, however, does not apply to the aminopeptidase cathepsin H. Screening of propeptide fragments for their inhibitory potency revealed no significant effect on the native mature cathepsin H. On the other hand, inhibitory interaction was established with recombinant cathepsin H that displays endopeptidase activity due to a lack of the mini-chain. This finding suggests that the propeptide-binding region is structurally rearranged during maturation processing and mini-chain formation, which impairs the effective recognition of mature cathepsin H by its own propeptide.