Experimental data required to support second medical use claims.

The timing of filing a new patent application always requires a judgment call to be made between securing the earliest filing date and waiting to collect enough data to ensure the claims are fully supported and enabled across their scope. In addition, it is important to ensure that the application is drafted in such a way that it is suitable for filing in various jurisdictions. The case law we have considered for the sufficiency/enablement of second medical use claims in Europe, the USA and Australia makes it clear that experimental results should be included in the application at filing, and that the results must demonstrate that the claimed pharmaceutical product could be reasonably expected to treat the claimed indication.

A novel drug discovery concept for tuberculosis: inhibition of bacterial and host cell signalling.

The Mycobacterium tuberculosis genome encodes for eleven eukaryotic-like Ser/Thr protein kinases. At least three of these (PknA, PknB and PknG) are essential for bacterial growth and survival. PknG is secreted by pathogenic mycobacteria, in macrophages to intervene with host cell signalling pathways and to block the fusion of the lysosomes with the phagosome by a still unknown mechanism. Based on our previously published results, we have initiated a drug discovery program, aiming to improve the potency against PknG and the physiochemical properties of the initially identified hit compound, AX20017, from the class of the tetrahydrobenzothiophenes. We have established a radioactive biochemical PknG kinase assay to test the novel analogues around AX20017. We have developed lead molecules with IC50 values in nanomolar range, and demonstrated their antituberculotic effects on human macrophages. Selected leads might ultimately serve the purpose of inducing phagosomal-lysosomal fusion and therefore destroy the residence of the intracellular mycobacteria. It is unclear at this time if these "homeless" mycobacteria are getting killed by the host, but they will be at least vulnerable to the activity of antimycobacterial agents. Released mycobacteria rely on the essential function of PknB for survival, which is our second molecular kinase target. PknB is a transmembrane protein, responsible for the cell growth and morphology. We have screened our library and synthesized novel compounds for the inhibition of PknB. A pharmacophore model was built and 70,000 molecules from our synthesizable virtual library have been screened to identify novel inhibitor scaffolds for the generation of templated compound libraries. Currently, we are using a radioactive kinase assay employing GarA as the putative, physiological substrate of PknB kinase. We have identified hits and generated optimised hit compounds with IC50 values for the inhibition of PknB in the nanomolar range. Yet those promising hits are not potent enough to yield meaningful "minimum inhibitory concentrations" in mycobacterial growth assays. In the course of our future work, we will increase the potency of the next generation of PknB inhibitors in order to improve their antibacterial activity.

Learning opportunities for Australian prevocational hospital doctors: exposure, perceived quality and desired methods of learning.

OBJECTIVE: To survey prevocational doctors working in Australian hospitals on aspects of postgraduate learning. PARTICIPANTS AND SETTING: 470 prevocational doctors in 36 health services in Australia, August 2003 to October 2004. DESIGN: Cross-sectional cohort survey with a mix of ordinal multicategory questions and free text. MAIN OUTCOME MEASURES: Perceived preparedness for aspects of clinical practice; perceptions of the quantity and usefulness of current teaching and learning methods and desired future exposure to learning methods. RESULTS: 64% (299/467) of responding doctors felt generally prepared for their job, 91% (425/469) felt prepared for dealing with patients, and 70% (325/467) for dealing with relatives. A minority felt prepared for medicolegal problems (23%, 106/468), clinical emergencies (31%, 146/469), choosing a career (40%, 188/468), or performing procedures (45%, 213/469). Adequate contact with registrars was reported by 90% (418/465) and adequate contact with consultants by 56% (257/466); 20% (94/467) reported exposure to clinical skills training and 11% (38/356) to high-fidelity simulation. Informal registrar contact was described as useful or very useful by 94% (433/463), and high-fidelity simulation by 83% (179/216). Most prevocational doctors would prefer more formal instruction from their registrars (84%, 383/456) and consultants (81%, 362/447); 84% (265/316) want increased exposure to high-fidelity simulation and 81% (283/350) to professional college tutorials. CONCLUSION: Our findings should assist planning and development of training programs for prevocational doctors in Australian hospitals.

Structure of glyceraldehyde-3-phosphate dehydrogenase from Plasmodium falciparum.

The malaria parasite Plasmodium falciparum is responsible for about two million deaths annually, making it important to obtain information about enzymes from this organism that represent potential drug targets. The gene for P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH) has been cloned and the protein expressed as a hexahistidine-tagged recombinant protein in Escherichia coli. The recombinant protein has been crystallized and its three-dimensional structure determined. One molecule of the cofactor NAD+ is bound to each of the four subunits in the tetrameric enzyme. The major structural feature distinguishing human GAPDH from PfGAPDH is the insertion of a dipeptide (-KG-) in the so-called S loop. This insert, together with other characteristic single-amino-acid substitutions, alters the chemical environment of the groove that encompasses the R dyad and that links adjacent cofactor-binding sites and may be responsible for the selective inhibition of the enzyme by ferriprotoporphyrin IX.

Cloning of the Pneumocystis jirovecii trifunctional FAS gene and complementation of its DHPS activity in Escherichia coli.

Pneumocystis pneumonia or PCP is caused by Pneumocystis jirovecii, an obligate parasite of the human lung. In this study P. jirovecii genomic sequence encoding FAS, a trifunctional protein including dihydroneopterin aldolase (DHNA), hydroxymethyldihydropterin pyrophosphokinase (PPPK) and dihydropteroate synthase (DHPS) were identified by PCR amplification from fixed broncheolar lavage samples from patients having Pneumocystis pneumonia. The P. jirovecii trifunctional DHNA-PPPK-DHPS genes (PjFAS) showed a high degree of conservation with the rat Pneumocystis carinii and P. carinii f. sp. macaca sequences. To test the functionality of the PjFAS sequences introns were removed followed by cloning and expression of PjFAS sequences in a DHPS-disrupted Escherichia coli strain. Complementation depended on the presence of N-terminal FAS sequences in addition to a glutathione S- transferase tag to the N-terminus of PjFAS. Functional complementation allowed evaluation of DHPS mutations implicated with sulfa drug resistance.

Inhibition studies of sulfonamide-containing folate analogs in yeast.

In the folate biosynthetic pathway, sulfa drugs (sulfonamides and sulfones) compete with the natural substrate, para-aminobenzoate (pABA) causing depletion of dihydrofolate (DHF) and subsequent growth inhibition. The sulfa drugs condense with 2-amino-4-hydroxy-6-hydroxymethyl-7,8 dihydropteridine pyrophosphate (DHPPP) forming sulfa-dihydropteroate (sulfa-DHP). Here evidence is presented using yeast that such dihydropteroate (DHP) analogs are inhibitory through competition with DHF. Two folate synthesis mutants, with respective dihydrofolate synthase (DHFS) and dihydropteroate synthase (DHPS) deletions and requiring DHF for growth were exposed to sulfa drugs. The DHFS knockout mutant was inhibited, but the DHPS knockout mutant that was incapable of forming sulfa-DHP was insensitive. Such sulfa-DHP compounds were chemically synthesized and shown to be inhibitory in vivo by competing with DHF, but in vitro assays with double the concentration of the sulfa-DHP to DHF showed no inhibition of dihydrofolate reductase (DHFR). Sequence analysis of resistant mutants obtained in the presence of sulfa drugs showed no changes in DHFR, or DHPS, unlike previously found antifolate-resistant mutants. The diamino derivatives, which are precursors of the sulfa-DHP, were found to be DHFR inhibitors. These results suggest that a new class of drugs, based on DHP analogs, could be investigated.