WH – Page 9 – Drug Discovery Opinion

Structure of “Caffeine Receptor” Solved

Collaborating scientists at the Scripps Research Institute and The Amsterdam Center for Drug Research have determined the crystal structure of the human adenosine A_2A receptor, also known as the caffeine receptor. The receptor is a member of the hetero-trimeric G-protein coupled receptor (GPCR) superfamily and plays an important role in mediating responses to adenosine in many physiological processes. The scientists were able to obtain crystals of the protein by binding it to a potent adenosine antagonist, ZM241385, which had been developed as a potential drug to combat Parkinson’s disease. Full details have been published in the journal Science.

Despite the importance of GPCRs as drug targets, determination of their crystallographic structure has proven difficult. This new structure follows the success of the Scripps team’s publication of the β2-adrenergic receptor structure last year.

Adenosine interacts with a number of GPCRs including the A₁, A_2A, A_2B, and A₃ subtypes. Each of these plays a role in responding to adenosine in the central nervous system in pain regulation, cerebral blood flow, basal ganglia functions, respiration, and sleep. Insights obtained from the study of the A_2A structure have already suggested mechanisms for receptor subtype selectivity.

It is hoped that this new information will help in the design of new drugs that could be important in the treatment of numerous neurological disorders, including Parkinson’s and Huntington disease.

Chemical Biology used to Probe Apoptotic Pathways

The journal Nature Chemical Biology defines chemical biology as ‘both the use of chemistry to advance a molecular understanding of biology and the harnessing of biology to advance chemistry’.

Scientists at the Karolinska Institute, Stockholm, have exploited the principles of chemical biology in the study of apoptotic pathways. In a recent publication, the scientists identified a set of 40 chemical agents (‘bioprobes’) that induce apoptosis from screening of a chemical library.

helenalin Using a variety of reporter cell lines, they were able to establish that the ‘bioprobes’ induced different patterns of signalling. Experiments using a calcium chelator, BAPTAAM, showed that Ca²⁺ was involved in induction of apoptosis by the majority of the ‘bioprobes’ and that Ca²⁺ was in general required several hours into the apoptosis process. Further studies showed that the calmodulin pathway was an important mediator of the apoptotic response. Inhibition of calmodulin kinase II (CaMKII) resulted in more effective inhibition of apoptosis compared to inhibition of calpain, calcineurin/PP2B or DAP kinase. One of the ‘bioprobes’, the plant alkaloid helenalin, was used to study the role of CaMKII in apoptosis. Helenalin induced CaMKII, ASK1 and Jun-N-terminal kinase (JNK) activity, and inhibition of these kinases inhibited apoptosis.

The study shows that calcium signalling is generally not an early event during the apoptosis process and suggests that a CaMKII/ASK1 signalling mechanism is important for sustained JNK activation and apoptosis by some types of stimuli.

Rheb is Overexpressed in Certain Cancers

The mTOR (mammalian target of rapamycin) pathway represents a convergence point for signalling pathways commonly disrupted in cancer. The pathway includes several known and putative oncogenes as well as tumour suppressors. Rheb GTPase is the upstream activator of the mTOR Complex 1 (mTORC1) and is itself activated by growth factors and nutrients.

Two independent papers in the August 15th issue of Genes and Development link Rheb activity with particular cancers. Wendel et. al. demonstrate that Rheb activation can produce rapid development of aggressive and drug-resistant lymphomas. The authors further show that activation of mTORC1 is dependent on farnesylation of Rheb and that an inhibitor of farnesyl transferase (FTI) is able to block the activation. It is noted that Rheb is highly expressed in certain human lymphomas.

Pandolfi et. al. show that overexpression of Rheb promotes hyperplasia and a low-grade neoplastic phenotype in the mouse prostate. Additionally, Rheb overexpression combined with Pten haploinsufficiency results in marked promotion of prostate tumorigenesis.

These results suggest potential for Rheb as a therapeutic target in particular oncology indications.

New Approach to Treatment of Schizophrenia

Schizophrenia is a complex disorder, generally believed to arise from dysregulation of dopamine and glutamate neurotransmission pathways. Current front-line treatment comprises the atypical antipsychotics, which provide symptomatic relief but are associated with significant side-effects. These agents are broad-spectrum GPCR antagonists that act primarily at the dopamine and serotonin receptors.

Muscarinic acetylcholine (mACh) receptors regulate dopamine levels in areas of the brain associated with psychosis, with the M₄ subtype speculated to be a key regulator of dopaminergic hyperactivity. The absence of subtype-selective modulators has, however, hindered validation of this hypothesis. Now scientists at Lilly have reported (PNAS, 5th August 2008) a selective small molecule, LY2033298, that targets the M₄ subtype.

LY2033298 The compound has been shown to act at an allosteric site on the receptor, potentiating agonist binding while having little effect on antagonist binding. The authors have further demonstrated in vivo activity in preclinical models that are predictive of antipsychotic drug effects.

Progress in Targeting the Sliding Clamp

There has been interest in the DNA polymerase sliding clamp as an antibacterial target for the last 15 years. Sliding clamp proteins, found in all organisms, encircle DNA (and slide along it!) and tether polymerases to enable rapid and processive DNA replication. The proteins are known as proliferating cell nuclear antigen (PCNA) in eukaryotes and as the β-clamp in prokaryotes. In PCNA the clamp is composed of three subunits of two domains each, whilst the bacterial β-clamp is assembled from two subunits of three domains. Although the overall structures of the eukaryotic and prokaryotic clamps are similar, there is no detectable sequence homology.

RU7 In a paper to be published in the August 12^th edition of PNAS, the authors disclose a small molecule inhibitor of the E.coli β-clamp, RU7, which differentially inhibits polymerases II, III and IV.

RU7 selectively inhibits Pol III in β-dependent replication assays, with no activity in the eukaryotic PCNA system. The compound, which has modest potency, was identified by screening for compounds able to displace a Pol III peptide from the β-clamp. The authors have also determined the co-crystal structure of RU7 bound to the clamp (pdb identifier 3d1g), paving the way for structure-based design.
PDB:3D1G

PDB:3D1G

Plk1 in Oncology – the Case Strengthens

Polo-like kinase 1 (Plk1) has received attention in recent years as a potential target for intervention in cancer. It is known to be important in regulation of cell cycle progression during M-phase and has been shown to be overexpressed in certain tumours. Now scientists at the NYU Cancer Institute and Howard Hughes Medical Institute have shown that Plk1 is involved in a new pathway associated with the cellular response to DNA damage. In the July 25th issue of Cell, the authors describe targeting of the phosphatase Cdc14B to APC/C, a protein that marks other proteins for destruction. The resultant activated APC/C then tags Plk1 for destruction. If Plk1 remains active, the cell continues to divide despite the DNA damage.

Tekmira Pharmaceuticals, a specialist in delivery of RNA interference molecules, has just announced plans to advance an siRNA product targeting Plk1 into Phase 1 clinical trials in the second half of 2009. Meanwhile, the search for small molecule inhibitors of Plk1 continues. Scientists at Pfizer deposited x-ray coordinates of the catalytic domain of a mutant Plk1(complexed with BI2536) in the protein data bank earlier this year. Additional structure factors have been deposited by Sunesis scientists, although the coordinates have not yet been released.