The increased understanding of underlying neurobiological processes and their connections to cognitive performance has opened new doors for novel therapeutic strategies that aim to prevent, and even reverse the effects of memory disorders, neurodegenerative diseases and other cognitive impairments. One of the new and exciting drug targets that has emerged as a result of this research is insulin-regulated aminopeptidase (IRAP). While the research is still in its early stages, inhibition of IRAP have promising therapeutic potential and several small molecule and peptidomimetic candidates that act as inhibitors of IRAP have been synthesized. Analysis of in vivo performed novel object recognition tests, in vitro cell viability and morphology assays were conducted in this study to further investigate the cognitive effects and other effects of an IRAP-inhibitor called HA08. Recorded novel object recognition test previously performed on rats were scored and analysed with statistical software. Primary cortical and hippocampal cells harvested from rat fetuses were treated with increasing concentrations of HA08 and oxytocin. The cell viability assays consisted of LDH assays to analyse HA08’s effect on the cells membrane integrity and MTT assays to analyse its effect on mitochondrial function. Immunocytochemistry was used to assess the difference in neurite length in cortical cells and the number of dendritic spines in hippocampal cells. No increases in recognition were found in HA08 treated rats. HA08 did not seem to affect the membrane integrity at concentrations used. Further research is needed to improve the accuracy of the results and to fully unlock the potential of IRAP-inhibitor HA08.

The need for alternative cognitive enhancers has risen due to the fact that clinical trial results of the drugs currently approved for treating these disorders have not been satisfactory. IRAP has become a possible drug target for treating cognitive impairment brought about by Alzheimer’s disease, head trauma or cerebral ischemia, among others. This came after the revelation that Angiotensin IV enhances memory and learning. Angiotensin IV, the endogenous ligand of IRAP has been structurally modified with the aim of producing potent IRAP inhibitors. However, the peptidic nature of these inhibitors restricts their use; they are not likely to cross the blood brain barrier. Other strategies for generating IRAP inhibitors have been through structure-based design and receptor based virtual screening. These drug-like molecules have exhibited positive results in animal studies. IRAP inhibitors have been identified via a HTS of 10500 low-molecular weight compounds to give the hit based on a spirooxindole dihydroquinazolinone scaffold, with an IC50 value of 1.5 µM. In this project, some analogues to this hit compound have successfully been synthesized using a known method, whereas others have been synthesized after additional method development. The application of the developed method was found to be limited, because poor yield was obtained when a compound with an electron withdrawing substituent on the aniline was synthesized. As a result of this, modification of this method may be required or new methods may have to be developed to synthesize these types of analogues. Inhibition capability of 5 new spirooxindole dihydroquinazolinones was tested through a biochemical assay. Compound 6e emerged as the most potent inhibitor in the series, with an IC50 value of 0.2 µM. This compound will now serve as a lead compound and should be used as a starting point for future optimization in order to generate more potent IRAP inhibitors.

Insulin regulated aminopeptidase (IRAP) was first identified in fat and muscle cells where it is believed to regulate GLUT4 translocation. It has since been found to be behind a variety of functions, many not yet fully understood. Preliminary research from Monash University suggested that IRAP may play a role in cellular senescence. Senescence is a term that describes arrested cell division and is a tumor repressive mechanism. Senescent cells have been shown to secrete, among other things, the growth hormone TGFβ1, which in turn plays an important role in the cell differentiation of fibroblasts to myofibroblasts. The potential link between IRAP and senescence was the basis of this work. Senescent fibroblasts from three different passages (n=3) in the BJ3 cell-line were cultured and treated with different IRAP inhibitors; ANG-4, AL06 and HFI-419 which were all compared to an untreated control group. They were marked with a β-galactosidase stain, a senescent cellmarker, and imaged. The study demonstrated that the IRAP inhibitors led to a certain decrease in % of senescent cells compared to the control groups. However, this reduction was not considered statistically significant. Similarly, inhibition of the enzyme did not indicate any influence over the differentiation of the cells. The lack of effect could be due to chance based on the low number of sample size, or the condition of the cells used in the trial as they were partially immortalized BJ fibroblasts well beyond the passage of their intended use. In order to further demonstrate an association between IRAP and senescence, further trials are required. / Insulin reglerad aminopeptidas (IRAP) introducerades till en början som ett markörprotein. Man har sedan dess funnit att den står bakom en rad olika funktioner, många ännu inte  fullt klarlagda. Preliminär forskning från laboratoriet i Monash University tydde på att IRAP kan ha en koppling till senescerande fibroblaster. Senescence är en term som beskriver upphörd celldelning och är en tumörrepressiv mekanism. Senescerande celler har påvisats utsekrera bland annat tillväxthormonet TGFβ1, som i sin tur spelar en viktig roll i celldifferentieringenav fibroblaster till myofibroblaster. Den potentiella kopplingen mellan IRAP och senescence låg som grund till detta arbete. Senescerande fibroblaster från tre olika kulturer (n=3) i BJ3-cellinjen odlades och behandlades med olika IRAP-inhibitorer; ANG-4, AL06 och HFI-419 som alla jämfördes med en kontrollgrupp. Därefter markerades de med en β-galaktosidas-markör, en markör för senescerande celler, och mikroskoperades. Studien påvisade att IRAP-inhibitorerna ledde till en viss procentuell minskning av senescerande celler jämfört med kontrollgrupperna. Dock bedömdes inte denna minskning som statistiskt signifikant i studien. Likväl fann man ingen procentuell minskning av differentierade fibroblaster. Hypotetiskt sett skulle man vilja se att reduktionen av senescerande celler motsvarade en nedreglering av TGFβ1-proteiner. Eftersom närvaron av TGFβ1 tros spela en ledande roll i celldifferentiering till myofibroblastfenotypen, bör den procentuella mängden differentierade cellerna minska med inhibitorbehandlingarna. Den bristande påverkan av enzyminhibitionen kan bero på en rad olika faktorer. Cellerna som användes under försökets gång var väl bortom deras brukliga användningscykel. För att vidare påvisa ett potentiellt samband mellan IRAP och senescence behöver vidare försök utföras.