Humans have a range of proteases, which are important for a range of processes including digestion of food, blood clotting, control of blood pressure, etc.  However, there are specific proteases, which can be targeted to treat various diseases, e.g. the AIDS virus needs a protease to multiply, cancers and parasites use proteases to move through tissues, proteases are used to produce the amyloid plaque protein which causes Alzheimer's disease.  Therefore, to treat such conditions we need to inactivate the proteases, which are required for the disease to progress.  This inactivation can be achieved using protease inhibitors.  

There are four main types of proteases: the thiol proteases, the serine proteases, the metalloproteases and the aspartyl proteases.  We usually wish to target just the one protease causing the disease and not inhibit other proteases, which are essential for health. We are synthesising protease inhibitors and using NMR to determine how they interact with specific proteases.  These studies will help us optimise their ability to inhibit the specific proteases involved in a range of diseases.

Our earlier studies utilised substrate derived chloromethylketone inhibitors, which alkylated the active site histidine and formed tetrahedral adducts analogous to the tetrahedral intermediate formed during catalysis. From these studies, we could quantify oxyanion stabilisation in both chymotrypsin and subtilisiin[1]

We have now extended our studies to reversible glyoxal inhibitors. In substrate derived glyoxal inhibitors the peptide carboxylate group(-COOH) is converted into a glyoxal group(-COCHO). We have synthesized Z-Ala-Pro-Phe-glyoxal and have found that it is a good inhibitor of the serine proteases chymotrypsin[2] and subtilisin[4] with Ki values of 25 nM and 2.3 µM respectively.  Using NMR, we have shown that with both enzymes the inhibitors form hemiketal complexes, which mimic the tetrahedral intermediate formed during catalysis. We have also synthesized z-Phe-Ala-glyoxal which we have shown is an extremely effective inhibitor (Ki =3.3 nM)= of papain [3]. Using NMR, we show that the active site thiol group of papain reacts with the glyoxal aldehyde carbon to form a thiohemiacetal. 

We have shown that Z-Ala-Ala-Phe-glyoxal is a potent inhibitor(Ki = 89 nM) of the aspartyl protease pepsin and using NMR we have found that the fully hydrated glyoxal inhibitor is bound to pepsin. We have also studyied the metalloproteinases thermolysin and stromelysin.

We are currently developing new glyoxal based inhibitors which can be used to inhibit falcipain 3 and urokinase plasminogen activator(uPA). It is hoped that these studies will help in the development of drugs to treat conditions such as cancer and malaria. We are also studying the mechanism of inhibition and we are developing methods for quantifying how inhibitor warheads such as the glyoxal group contribute to inhibitor potency in proteases.

• Mr. Edward Spink
• Ms. Máire Ní Beilliú 
• Ms. Sonya Cosgrove
• Ms. Nicole Howe
• Mr. Iñigo Alaña
• Ms. Mariangela Ceruso 
   
  

Inhibition of proteases (Collaborator Dr Paul Evans)

Using NMR to follow metabolism in cells (Collaborators: Drs Newsholme, McBean,Dr Brennan UCD)

Metabolomics (Collaborator: Dr Brennan, UCD)

Professor Hideaki Nagase, Imperial College London, UK

Professor W.W. Bachovchin, Tufts University,USA

Professor Qun, Peking University, China 

Dr N. O'Donovan, Dublin City University, Ireland 

Professor Joe Duffy, UCD

Professor P Rosenthal, Dept.Medicne, UCSF, USA

Biochemistry NMR centre
Conway Institue of Biomedical and Biololecular Research, UCD
http://www.ucd.ie/bio-nmr/Research1.html