Human cathepsin D (CatD), a pepsin-family aspartic protease, plays an important role in tumor progression and metastasis. Here, we report the development of biomimetic inhibitors of CatD as novel tools for regulation of this therapeutic target. We designed a macrocyclic scaffold to mimic the spatial conformation of the minimal pseudo-dipeptide binding motif of pepstatin A, a microbial oligopeptide inhibitor, in the CatD active site. A library of more than 30 macrocyclic peptidomimetic inhibitors was employed for scaffold optimization, mapping of subsite interactions, and profiling of inhibitor selectivity. Furthermore, we solved high-resolution crystal structures of three macrocyclic inhibitors with low nanomolar or subnanomolar potency in complex with CatD and determined their binding mode using quantum chemical calculations. The study provides a new structural template and functional profile that can be exploited for design of potential chemotherapeutics that specifically inhibit CatD and related aspartic proteases.

• MeSH
• biomimetické materiály chemická syntéza   chemie   metabolismus   toxicita   MeSH
• Caco-2 buňky MeSH
• cyklické peptidy chemická syntéza   chemie   metabolismus   toxicita   MeSH
• enzymatické testy MeSH
• inhibitory proteas chemická syntéza   chemie   metabolismus   toxicita   MeSH
• kathepsin D antagonisté a inhibitory   chemie   metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• molekulární struktura MeSH
• pepstatiny chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pepsin-family aspartic peptidases are biosynthesized as inactive zymogens in which the propeptide blocks the active site until its proteolytic removal upon enzyme activation. Here, we describe a novel dual regulatory function for the propeptide using a set of crystal structures of the parasite cathepsin D IrCD1. In the IrCD1 zymogen, intramolecular autoinhibition by the intact propeptide is mediated by an evolutionarily conserved exosite on the enzyme core. After activation, the mature enzyme employs the same exosite to rebind a small fragment derived from the cleaved propeptide. This fragment functions as an effective natural inhibitor of mature IrCD1 that operates in a pH-dependent manner through a unique allosteric inhibition mechanism. The study uncovers the propeptide-binding exosite as a target for the regulation of pepsin-family aspartic peptidases and defines the structural requirements for exosite inhibition.

• MeSH
• aktivace enzymů MeSH
• alosterická regulace MeSH
• katalytická doména MeSH
• kathepsin D chemie   metabolismus   MeSH
• kinetika MeSH
• klíšťata enzymologie   MeSH
• koncentrace vodíkových iontů MeSH
• krystalografie rentgenová MeSH
• ligandy MeSH
• peptidy chemie   metabolismus   MeSH
• prekurzory enzymů chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

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.

• MeSH
• apoptóza účinky léků   MeSH
• ceramidy chemie   metabolismus   farmakologie   MeSH
• fluorescenční polarizace MeSH
• fosforylace MeSH
• kathepsin D chemie   metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• nádory enzymologie   patologie   MeSH
• proteolýza účinky léků   MeSH
• rezonanční přenos fluorescenční energie MeSH
• sfingosin chemie   metabolismus   farmakologie   MeSH
• signální transdukce účinky léků   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Schistosomiasis caused by a parasitic blood fluke of the genus Schistosoma afflicts over 200 million people worldwide. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated peptidase that digests host blood proteins as a source of nutrients. It is under investigation as a drug target. To further this goal, we report three crystal structures of SmCB1 complexed with peptidomimetic inhibitors as follows: the epoxide CA074 at 1.3 Å resolution and the vinyl sulfones K11017 and K11777 at 1.8 and 2.5 Å resolutions, respectively. Interactions of the inhibitors with the subsites of the active-site cleft were evaluated by quantum chemical calculations. These data and inhibition profiling with a panel of vinyl sulfone derivatives identify key binding interactions and provide insight into the specificity of SmCB1 inhibition. Furthermore, hydrolysis profiling of SmCB1 using synthetic peptides and the natural substrate hemoglobin revealed that carboxydipeptidase activity predominates over endopeptidolysis, thereby demonstrating the contribution of the occluding loop that restricts access to the active-site cleft. Critically, the severity of phenotypes induced in the parasite by vinyl sulfone inhibitors correlated with enzyme inhibition, providing support that SmCB1 is a valuable drug target. The present structure and inhibitor interaction data provide a footing for the rational design of anti-schistosomal inhibitors.

• MeSH
• hemoglobiny chemie   MeSH
• hydrolýza účinky léků   MeSH
• inhibitory proteas chemie   MeSH
• kathepsin D antagonisté a inhibitory   chemie   genetika   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• peptidomimetika chemie   MeSH
• peptidy chemie   MeSH
• proteiny červů antagonisté a inhibitory   chemie   genetika   MeSH
• Schistosoma mansoni enzymologie   genetika   MeSH
• schistosomiasis mansoni farmakoterapie   enzymologie   genetika   MeSH
• systémy cílené aplikace léků MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

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.

• MeSH
• aminokyselinové motivy MeSH
• financování organizované MeSH
• glykosaminoglykany metabolismus   MeSH
• inhibitory proteas chemická syntéza   metabolismus   MeSH
• katalytická doména MeSH
• kathepsin D antagonisté a inhibitory   chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• peptidové fragmenty antagonisté a inhibitory   chemická syntéza   metabolismus   MeSH
• peptidové mapování MeSH
• prekurzory enzymů antagonisté a inhibitory   chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH

Cathepsin D is peptidase belonging to the family of aspartic peptidases. Its mostly described function is intracellular catabolism in lysosomal compartments, other physiological effect include hormone and antigen processing. For almost two decades, there have been an increasing number of data describing additional roles imparted by cathepsin D and its pro-enzyme, resulting in cathepsin D being a specific biomarker of some diseases. These roles in pathological conditions, namely elevated levels in certain tumor tissues, seem to be connected to another, yet not fully understood functionality. However, despite numerous studies, the mechanisms of cathepsin D and its precursor's actions are still not completely understood. From results discussed in this article it might be concluded that cathepsin D in its zymogen status has additional function, which is rather dependent on a "ligand-like" function then on proteolytic activity.

• MeSH
• kathepsin D chemie   metabolismus   fyziologie   MeSH
• lidé MeSH
• nádory patofyziologie   MeSH
• prekurzory enzymů metabolismus   fyziologie   MeSH
• proliferace buněk MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH