New Malaria Target: Blocking Protein Transport

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Image: Flickr - Marc van der Chijs
The lifecycle of all Plasmodium species is complex and involves a round of replication in host erythrocytes. The clinical manifestations of malaria are linked to this stage in the lifecycle and are associated with rupture of the infected erythrocytes. During this growth phase, the parasite enters the erythrocyte and then releases several hundred effector proteins into the cytoplasm. These key virulence proteins provide a suitable environment for multiplication and allow the parasite to evade the host immune system. Proteins destined for export contain a conserved pentameric motif known as PEXEL and, when this is cleaved in the endoplasmic reticulum, the protein can be transported into the host cell. Two independent studies by scientists in the US and Australia have now shown that the protease responsible for cleaving the PEXEL motif is the aspartyl protease, plasmepsin V. Cleavage reveals an export signal at the amino terminus of the cargo protein which is then transported into the host cell cytoplasm, likely through a channel in the parasite’s outer membrane. Since export of the effector proteins is essential for the erythrocytic stage of the plasmodium life-cycle, drugs that block plasmepsin V should provide an effective treatment for malaria.

Both studies are published in the journal Nature (Australian study; US study).

Battle of the Bulge

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Image: Flickr – zappowbang
An aortic aneurysm is a bulge in the aorta, the largest blood vessel in the body, which results from weakening of the artery wall. The majority of these occur in the portion of the aorta that passes through the abdomen and are referred to as abdominal aortic aneurysms (AAA). AAA is something of a stealth disease, since it is generally asymptomatic and may only be diagnosed at a routine physical examination or following X-ray. Over time the aneurysm may expand, with an increased risk of rupturing. Unfortunately, the rapid blood loss following aneurysm rupture is frequently fatal and accounts for at least 15,000 deaths in the US annually.

The only treatment for AAA currently available is surgical intervention. Early diagnosis is followed by monitoring the size of the aneurysm until the risk of rupture exceeds the risk of surgery. However, scientists at Providence Heart + Lung Institute at St. Paul’s Hospital and the University of British Columbia (UBC) have now raised the possibility of pharmacological intervention. Using experimental models of AAA, the team have found a role for the protein-degrading enzyme Granzyme B (GMZB).

GMZB is a serine protease expressed by a variety of immune cells and is responsible for destroying unwanted tissue, such as virally-infected cells. This role is supported by the pore-forming protein, perforin, which delivers GMZB to the intracellular compartment. The UBC research has shown that GMZB, which is abundantly expressed in aneurysms from human and animal model AAA, also plays a role in the pathogenesis of AAA. Further, the experimental data suggest that this is a perforin-independent mechanism involving extracellular matrix degradation and subsequent loss of vessel wall integrity. The results suggest that an inhibitor of GMZB may provide a therapeutic option in the treatment of AAA.

The study is published in the American Journal of Pathology.

Protease Inhibitor Treats Rare Genetic Brain Disorder

Lissencephaly (literally “smooth brain”) describes a set of rare conditions in which the foetal brain does not develop normally beyond the third or fourth month of pregnancy and, instead of the usual folds and grooves, the cerebrum has a partially or completely smooth appearance. The severity and range of symptoms varies, but include mental retardation, failure to thrive, difficulty swallowing, seizures, and psychomotor problems. A number of factors are believed to cause lissencephaly including viral infection during pregnancy, an interrupted blood supply to the foetus, and genetic mutation. Loss of one copy of the gene LIS1 prevents migration of immature nerve cells from deep in the brain to the surface of the emerging cerebral cortex and US and Japanese researchers have now shown, in mice at least, that the results of this mutation can be reversed during pregnancy, leading to more normal offspring.

human brainIn mice with only one copy of the LIS1 gene, the enzyme calpain reduces LIS1 protein levels to less than half of normal near the surface of cells, leading to abnormal brain development similar to that seen in human lissencephaly. Daily intraperitoneal injections of the small molecule calpain inhibitor, ALLN (N-Acetyl-Leu-Leu-Nle-CHO), to pregnant mice restored levels of LIS1 protein and resulted in offspring with more normal brains and no signs of mental retardation. Although the technique will not be easy to extend to humans, this study is the first successful attempt to use a protease inhibitor to reverse a severe brain defect caused by a partial deficiency in one key gene, and offers a proof-of-principle that the genetic equivalent to human lissencephaly can be effectively reversed during pregnancy to produce more normal offspring.

The authors hope that the approach could be extended to in utero treatments for other defects in which a protease plays a role in degrading an essential developmental protein.

The study was published online on September 6th in the journal Nature Medicine.

Small Molecule Inhibitors of Kaposi’s Sarcoma Virus Protease

Kaposi’s sarcoma (KS) is caused by Kaposi sarcoma herpes virus (KSHV) which is also known as human herpes virus 8 (HHV8). HHV8 infection rates vary widely amongst different populations but KS rarely develops unless the immune system is compromised, by AIDS, transplant drugs, or ageing. kaposi's sarcomaThere is currently no specific treatment for KS but researchers at UCSF have now identified small molecules that target the viral protease. Along with other members of the herpes family of viruses – including herpes simplex viruses I and II, varicella zoster virus, cytomegalovirus and Epstein-Barr virus – HHV8 encodes a serine protease that is essential for viral capsid formation and viral replication. Many previous attempts to discover inhibitors of herpes virus proteases targeting the active site of the enzymes met with limited success, perhaps because of difficulty in finding molecules that bind tightly to the shallow substrate-binding cleft. The UCSF team have chosen instead to inhibit catalytic activity by disrupting dimerisation of the enzyme.

A number of earlier studies have shown that dimerisation is a common mechanism for activation of herpes virus proteases, and the UCSF team have previously identified a helical peptide that prevents dimerisation of herpes virus proteases. In the new study, published online in the journal Nature Chemical Biology, the team describe small molecules, DD2 structureincluding DD2, which inhibit dimerisation of both HHV8 and CMV proteases with IC50s in the low micromolar range.

HIV protease also acts as an obligate dimer but, in this case, dimerisation inhibitors have been less successful than compounds that directly target the active site, many of which are now in clinical use. The difficulties experienced in trying to identify active site inhibitors of the herpes virus serine proteases may mean that disruption of dimerisation presents a more attractive target for these challenging enzymes.

Protease Inhibitors Could Be Effective For Fungal Disease

chromoblastomycosisChromoblastomycosis is a chronic fungal infection of the skin and subcutaneous tissue most commonly caused by infection with Fonsecaea pedrosoi. The disease occurs most often in rural areas in tropical and subtropical countries and is caused when fungus is introduced by minor injury such as that caused by a splinter or thorn. Chromoblastomycosis is very difficult to cure; antifungal chemotherapy, surgical excision and/or cryosurgery have traditionally been used but with varying degrees of success.

Recently, HIV protease inhibitors have been shown to have a direct effect on AIDS-related opportunistic pathogens such as Candida albicans by inhibiting production of C. albicans secreted aspartyl proteases. The proteases assist the fungus to colonize tissues and to evade the host’s antimicrobial defense mechanisms. F. Pedrosoi also secretes aspartyl proteases and a study published in PloS ONE describes the effect of selected HIV protease inhibitors on secreted protease activity and survival of F. Pedrosoi. At high concentration (100µM), saquinavir and nelfinavir robustly inhibited growth of F. Pedrosoi in vitro. The high concentration needed possibly reflects a much lower affinity for the fungal protease than for HIV protease, or may suggest alternative mechanisms of control. The authors also studied the in vitro effect of combining sub-inhibitory concentrations of the aspartyl protease inhibitors with sub-inhibitory concentrations of antifungal drugs and found good synergistic actions.

The results suggest that combination therapy with protease inhibitors and antimycotic drugs may be effective for treatment of chromoblastomycosis, especially if more potent inhibitors of the F. Pedrosoi aspartyl protease were developed.