Thioamide-based fluorescent protease sensors.

Thioamide quenchers can be paired with compact fluorophores to design "turn-on" fluorescent protease substrates. We have used this method to study a variety of serine-, cysteine-, carboxyl-, and metallo-proteases, including trypsin, chymotrypsin, pepsin, thermolysin, papain, and calpain. Since thioamides quench some fluorophores red-shifted from those naturally occurring in proteins, this technique can be used for real time monitoring of protease activity in crude preparations of virtually any protease.

A host GPCR signaling network required for the cytolysis of infected cells facilitates release of apicomplexan parasites.

Following intracellular replication, the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii cause host cell cytolysis to facilitate parasite release and disease progression. Parasite exit from infected cells requires the interplay of parasite-derived proteins and host actin cytoskeletal changes; however, the host proteins underlying these changes remain obscure. We report the identification of a Gα(q)-coupled host-signaling cascade required for the egress of both P.

Platelet factor 4 activity against P. falciparum and its translation to nonpeptidic mimics as antimalarials.

Plasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV).

Targeting the ERAD pathway via inhibition of signal peptide peptidase for antiparasitic therapeutic design.

Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells, and, because of this, we observe that the malaria parasite Plasmodium falciparum is highly sensitive to the inhibition of components of this protein quality control system. Inhibitors that specifically target a putative protease component of ERAD, signal peptide peptidase (SPP), have high selectivity and potency for P.

Development of α-helical calpain probes by mimicking a natural protein-protein interaction.

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices.

OspC is potent plasminogen receptor on surface of Borrelia burgdorferi.

Host-derived proteases are crucial for the successful infection of vertebrates by several pathogens, including the Lyme disease spirochete bacterium, Borrelia burgdorferi. B. burgdorferi must traverse tissue barriers in the tick vector during transmission to the host and during dissemination within the host, and it must disrupt immune challenges to successfully complete its infectious cycle.

The Trypanosoma cruzi protease cruzain mediates immune evasion.

Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy.

The malaria parasite progressively dismantles the host erythrocyte cytoskeleton for efficient egress.

Plasmodium falciparum is an obligate intracellular pathogen responsible for worldwide morbidity and mortality. This parasite establishes a parasitophorous vacuole within infected red blood cells wherein it differentiates into multiple daughter cells that must rupture their host cells to continue another infectious cycle. Using atomic force microscopy, we establish that progressive macrostructural changes occur to the host cell cytoskeleton during the last 15 h of the erythrocytic life cycle.

Substrate specificity of Staphylococcus aureus cysteine proteases--Staphopains A, B and C.

Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function.

Synthesis of new (-)-bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase.

The malarial PfA-M1 metallo-aminopeptidase is considered a putative drug target. The natural product dipeptide mimetic, bestatin, is a potent inhibitor of PfA-M1. Herein we present a new, efficient, and high-yielding protocol for the synthesis of bestatin derivatives from natural and unnatural N-Boc-d-amino acids.

Discovery of potent small-molecule inhibitors of multidrug-resistant Plasmodium falciparum using a novel miniaturized high-throughput luciferase-based assay.

Malaria is a global health problem that causes significant mortality and morbidity, with more than 1 million deaths per year caused by Plasmodium falciparum. Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites, which necessitates the discovery of new drugs. To identify new antimalarials, we developed an automated 384-well plate screening assay using P.

A small-molecule oxocarbazate inhibitor of human cathepsin L blocks severe acute respiratory syndrome and ebola pseudotype virus infection into human embryonic kidney 293T cells.

A tetrahydroquinoline oxocarbazate (PubChem CID 23631927) was tested as an inhibitor of human cathepsin L (EC 3.4.22.

Signal peptide peptidases: a family of intramembrane-cleaving proteases that cleave type 2 transmembrane proteins.

Five genes encode the five human signal peptide peptidases (SPPs), which are intramembrane-cleaving aspartyl proteases (aspartyl I-CLiPs). SPPs have been conserved through evolution with family members found in higher eukaryotes, fungi, protozoa, arachea, and plants. SPPs are related to the presenilin family of aspartyl I-CLiPs but differ in several key aspects.

Apicomplexan parasites co-opt host calpains to facilitate their escape from infected cells.

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii (the causative agents of malaria and toxoplasmosis, respectively), are responsible for considerable morbidity and mortality worldwide. These pathogenic protozoa replicate within an intracellular vacuole inside of infected host cells, from which they must escape to initiate a new lytic cycle. By integrating cell biological, pharmacological, and genetic approaches, we provide evidence that both Plasmodium and Toxoplasma hijack host cell calpain proteases to facilitate parasite egress.

Development of bestatin-based activity-based probes for metallo-aminopeptidases.

A novel set of activity-based probes (ABPs) for functionally profiling metallo-aminopeptidases was synthesized based on the bestatin inhibitor scaffold, the first synthesis of bestatin analogues using solid-phase techniques. These ABPs were shown to label metallo-aminopeptidases, using both a biotin and a fluorophore reporter, in an activity-dependent manner. This probe class was also shown to be amenable to 'click' chemistry labeling for possible use in live cells.

Is chemical genetics the new frontier for malaria biology?

Malaria is a global disease, causing at least 500 million clinical cases and more than one million deaths each year. Moreover, drug-resistant Plasmodium falciparum, the organism that causes most malaria-associated deaths, has become a major problem. Therefore, discovery and investigation of novel targets for anti-malarial drug design is essential to combat this disease.

Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts.

Merozoite surface proteins of the human malaria parasite Plasmodium falciparum are involved in initial contact with target erythrocytes, a process that begins a cascade of events required for successful invasion of these cells. In order to identify complexes that may play a role in invasion we purified detergent-resistant membranes (DRMs), known to be enriched in merozoite surface proteins, and used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to isolate high molecular weight complexes for identification by mass spectrometry. Sixty-two proteins were detected and these mostly belonged to expected DRM proteins classes including GPI-anchored, multi-membrane spanning and rhoptry proteins.

Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities.

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids.

Distinct protein classes including novel merozoite surface antigens in Raft-like membranes of Plasmodium falciparum.

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol.

A cathepsin B-like protease is required for host protein degradation in Trypanosoma brucei.

Identification and analysis of Clan CA (papain) cysteine proteases in primitive protozoa and metazoa have suggested that this enzyme family is more diverse and biologically important than originally thought. The protozoan parasite Trypanosoma brucei is the etiological agent of African sleeping sickness. The cysteine protease activity of this organism is a validated drug target as first recognized by the killing of the parasite with the diazomethane inhibitor Z-Phe-Ala-CHN(2) (where Z is benzyloxycarbonyl).

Targeted disruption of Plasmodium falciparum cysteine protease, falcipain 1, reduces oocyst production, not erythrocytic stage growth.

Cysteine proteases are currently targets for drug development in a number of parasitic diseases, including malaria. In Plasmodium falciparum, the parasite responsible for the most virulent form of human malaria, there are four members of the cathepsin L-like family of cysteine proteases. Three of these (falcipains 2A, 2B and 3) are thought to be primarily involved in haemoglobin digestion, whereas falcipain 1 has recently been linked to erythrocyte invasion.

Synthesis and structure-activity relationships of parasiticidal thiosemicarbazone cysteine protease inhibitors against Plasmodium falciparum, Trypanosoma brucei, and Trypanosoma cruzi.

We have synthesized a library of thiosemicarbazones and screened them against three parasitic cysteine proteases, cruzain, falcipain-2, and rhodesain, and against the respective parasite sources of these three proteases, Trypanosoma cruzi, Plasmodium falciparum, and Trypanosoma brucei. The screens identified compounds that were effective against the enzymes and the parasites but also some compounds that were parasiticidal despite a lack of activity against the proteases. Several compounds were effective in killing all tested parasites.

Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis.

Tumors develop through successive stages characterized by changes in gene expression and protein function. Gene expression profiling of pancreatic islet tumors in a mouse model of cancer revealed upregulation of cathepsin cysteine proteases. Cathepsin activity was assessed using chemical probes allowing biochemical and in vivo imaging, revealing increased activity associated with the angiogenic vasculature and invasive fronts of carcinomas, and differential expression in immune, endothelial, and cancer cells.