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Journal of Proteomics & Bioinformatics - Open Access Research Article JPB/Vol.1/December 2008

Novel Therapeutic Approaches to Treat Alzheimer’s

Disease and Memory Disorders

David B. Ascher1, Gabriela A. N. Crespi1, Hooi Ling Ng1, Craig J. Morton1,

Michael W. Parker1,2* and Luke A. Miles1,2

1Biota Structural Biology Laboratory and Centre for Structural Neurobiology,St. Vincent’s Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065, Australia

2Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne,30 Flemington Road, Parkville, Victoria 3010, Australia

*Corresponding author: Prof. Michael W. Parker, Biota Structural Biology Laboratory,St. Vincent’s Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065, Australia

Tel: 0061 3 9288 2480; Fax: 0061 3 9416 2676; E-mail: [email protected]

Received August 09, 2008; Accepted October 14, 2008; Published December 05, 2008

Citation: David BA, Gabriela ANC, Hooi LN, Craig JM, Michael WP, et al. (2008) Novel Therapeutic Approaches to Treat Alzheimer’s Disease and Memory Disorders. J Proteomics Bioinform 1: 464-476. doi:10.4172/jpb.1000054

Copyright: © 2008 David BA, et al. This is an open-access article distributed under the terms of the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authorand source are credited.

Abstract

Cognitive decline most commonly associated with Alzheimer’s dementia can also result from other conditions

including cerebral ischemia or brain trauma. One quarter of people over the age of 65 are estimated to suffer

some form of cognitive impairment underscoring the need for effective classes of cognitive-enhancing agents. In

this mini-review we highlight recent work from our laboratory on the structural biology of Alzheimers disease

and other memory disorders that was presented at the recent PRICPS – AOHUPO 2008 Conference held in

Cairns, Australia. Our current work is focused on two proteins, amyloid precursor protein and insulin-regulated

aminopeptidase, that are promising targets for the development of anti-Alzheimer’s drugs and as cognitive en-

hancers. In both cases structures determined by X-ray crystallography are being used to discover promising

lead compounds by structure-based drug design.

Keywords: Alzheimer’s disease; Amyloid precursor protein; Drug design; Insulin-regulated aminopeptidase; X-ray crystal-lography

Abbreviations

Aβ: the peptide generated by cleavage of amyloid precursor protein; AD: Alzheimer’s disease; Ang IV: Angiotensin IV;AOHUPO: 4th Asian-Oceania Human Proteome Organization; APA: Aminopeptidase A; APB: Aminopeptidase B; APN:Aminopeptidase N; APP: Amyloid Precursor Protein; ColAP: Cold Aminopeptidase; ERAP: Endoplasmic Reticulum Aminopepti-dase; IRAP: Insulin-regulated Aminopeptidase; LTA4H: Leukotriene A4 hydrolase; PFA: Antibody that recognizes the Aβpeptide; PRICPS: 2nd Pacific Rim International Conference on Protein Science; TRDE: pyroglutamyl peptidase II; WO2:antibody that recognizes the Aβ peptide

Introduction

Approximately one quarter of people over the age of sixtyfive are estimated to suffer some form of cognitive impair-ment. The incidence of age-related neurological diseases isescalating primarily due to the increased life expectancy ofthe general population of many developed nations. One of

the more prevalent and debilitating neurological disorders isAlzheimer’s disease (AD).

AD is the leading cause of dementia among the elderlyand is characterized by the presence of amyloid plaques,

Full Paper Accepted from the Joint 2nd Pacific Rim International Conference on Protein Science and

4th Asian-Oceania Human Proteome Organization, Cairns- Australia, 22-26 June 2008


J Proteomics Bioinform Volume 1(9) : 464-476 (2008) - 465 ISSN:0974-276X JPB, an open access journal

extensive neuronal death and shrinkage of the brain. It isincreasingly accepted that the neurotoxic Ab peptide is re-sponsible for compromising neuronal functions and trigger-ing cell death (Selkoe, 2002; Pereira et al., 2004). The pep-tide is derived from the cleavage of the amyloid precursorprotein (APP) and is the main constituent of the amyloidplaques (Kang et al., 1987). Ab arises through the sequen-tial cleavage by the b-site APP cleaving enzyme (BACE) inthe ectodomain near the transmembrane domain and by theg-secretase protein complex within the membrane. Thisprocessing is thought to occur in endosomes or in an endo-some-trans golgi network recycling pathway. APP is a Type-I transmembrane protein with a large extracellular portion,which has been structurally and functionally subdivided intoseveral domains. We initiated a structural biology programfocused on APP about ten years ago with aim of determin-ing its structure as the basis for understanding its functionand for structure-guided drug design. The program has beenfruitful with numerous structures of the various APP do-mains now determined (Rossjohn et al., 1999; Barnham etal., 2003; Kong et al., 2005; Kong et al., 2007a; Kong etal., 2007b; Kong et al., 2008; Miles et al., 2008; Wun et al.,2008).

AD drug development strategies have focused on modu-lation of the Ab processing enzymes (β and γ secretases)and prevention of Aβ aggregation or oligomerization (Wolfe,2002). Currently all drugs approved by the US Food andDrug Administration for AD address disease symptoms.Most belong to the class of cholinesterase inhibitors, whichare of limited efficacy and only indicated for the treatmentof mild-to-moderate forms of the disease (Birks, 2006). Inspite of this, many drugs currently being developed to treatcognitive decline in AD are still targeting central cholinergicsystems (www.alzforum.org/drg/drc). Development ofmemory-enhancing drugs is gaining momentum because oftheir increasingly widespread application in the treatmentof other forms of memory disorders, including mild cogni-tive impairment, as well as that resulting from brain traumaand ischemic damage. Here we describe our recent studieson APP and on a protein receptor involved in memory.

Structural studies of an amyloid peptide-antibody

complex

One promising stream of research to develop treatmentsor prophylactics for AD is anti-amyloid-β (Aβ immuno-therapy) which aims to promote Aβ peptide clearance fromthe brain. In January 2002, however, the outlook for immu-notherapies to AD took a pessimistic turn when the firstclinical trials of active vaccination for AD were halted aftera small subset of patients developed sterile meningoencepha-

litis. The synthetic Aβ peptide antigen from those trials, AN-1792, is thought to have elicited a severe T-cell mediatedimmune response inducing a higher incidence of meningoen-cephalitis. Despite this setback, it was subsequently dem-onstrated that vaccination with AN-1792 elicited a positiveantibody response and that antibodies did not cross-reactwith APP as first feared. It was also shown that cognitivefunction was stabilised in over half of patients with highantibody titer (Weksler, 2004).

Six years later, Elan Corporation and Wyeth Pharmaceu-ticals, who initiated the AN-1792 trials, have begun a Phase3 clinical program of bapineuzumab (AAB-001), for the treat-ment of patients with mild to moderate Alzheimer’s disease.Bapineuzumab is a humanised monoclonal antibody target-ing Aβ and is the first antibody based therapy to AD toenter Phase 3 clinical trials. It is hoped that by taking apassive immunotherapy approach, detrimental immune re-sponses like those elicited by the AN-1792 immunogen canbe avoided.

Results of the small Phase 2 clinical trial of Bapineuzumabon 240 patients showed significant slowing in the rate ofmental decline and brain atrophy in some Alzheimer’s pa-tients receiving Bapineuzumab compared with placebo con-trol patients. Unfortunately, in the Phase 2 trials twelve pa-tients developed an accumulation of fluid on the brain knownas vasogenic edema. Vasogenic edema was shown to oc-cur at high doses and predominantly in patients carrying theE4 allele of apolipoprotein, a gene known to predispose acarrier to AD. With the incidence of vasogenic edema inmind, the Phase 3 trials initiated by Wyeth and Elan in 2007were designed to treat APO E4 carriers and non-

http://www.alzforum.org/new/detail.asp?id=1850). In addition to safety concerns raised inthe Phase 2 trials, efficacy trends were weaker than ex-pected. However, the much larger Phase 3 trials onBapineuzumab will establish the true efficacy of this par-ticular therapeutic in sufferers with mild to moderate formsof the disease.

A long term follow up study of the Phase 1 AN-1792 trialshowed that immunisation of patients with pre-fibrillated Aβ(AN-1792) initiated a long term reduction in Aβ load andpost mortem evidence of a plaque removal 5 years after thelast injection. (Holmes et al., 2008). In addition, the two pa-tients with almost complete elimination of plaques and low-est Aβ loads were shown to have had the highest antibodyresponse during the Phase 1 trial. Unfortunately, there wasno evidence that immunisation with AN-1792 had an im-pact on cognitive decline in the small cohort assessed in thefollow up study. It may be that immunotherapies targeting

carriers separately (



fibrillar Aβ (such as AN-1792) are not effective at reducingthe concentration of soluble oligomeric forms of the pep-tide, and it has been suggested that disintegration of plaquesmay increase the concentration of these oligomers impli-cated in synaptic dysfunction and dementia in Alzheimer’sdisease (Patton et al., 2006).

Antibodies raised against Aβ that have been shown toreduce plaque burden and improve cognitive deficits inmouse models of AD are those raised against the N-termi-nal region of Aβ (McLaurin et al., 2002; Frenkel et al.,2003; Chauhan and Siegel, 2005). Following the AN-1792 trials,development of passive and active immunotherapies tar-geting Aβ has focused on this N-terminal region (~ residues1 to 14), which contains the immunodominant B-cell epitopeof Aβ but lacks the T-cell epitopes arguably responsible forabandonment of the AN-1792 trials. A number of antibod-ies specific to this region have been studied for their abilitybind Aβ, inhibit fibrillogenisis and cytotoxicity, and reduceplaque burden in vivo (Solomon, 2004). In addition, severalengineered immunogens based on this B-cell epitope havebeen proposed as vaccine candidates. Considering the in-tense interest in antibody based therapies targeting Aβ, it issurprising that only recently has structural data emergedcharacterising the molecular basis for antibody recognitionof this important pathogen.

Crystal Structures

Concomitant structures of monoclonal antibody fragments

(Fabs) with specificity for the immunodominant B-cellepitope of Aβ have been reported for three Fabs inunliganded states and in complex with Aβ peptides. Dealwisand co-workers (Gardberg et al., 2007) reported high reso-lution 3D structures of Fabs from the monoclonal antibod-ies PFA1 and PFA2 in complex with the Aβ

1-8 peptide

(DAEFRHDS). We reported our own near-atomic resolu-tion structures (Miles et al., 2008) for the antigen bindingfragment of the WO2 antibody complexed to Aβ

1-16 and

Aβ1-28 peptides. Seven residues of Aβ (Ala 2 to Ser 8) havebeen modelled from electron density when complexed toPFA1, PFA2 or WO2. From our own work, extending thelength of the peptide from 16 to 28 residues yielded equiva-lent structures with no electron density observed beyondthis range, corresponding to peptide outside the restraints ofthe antigen binding site of WO2.

PFA1 and PFA2 have identical sequences across theirlight chains and indeed the WO2 light chain sequence isvery similar (95% sequence identity) to the PFA light chains.Across the heavy chains PFA1 and PFA2 sequences share96% sequence identity, whereas the WO2 heavy chain alignsto the PFA sequences with around 85% identity. The clearestpoint of difference is in the hypervariable CDR3 of the heavychains, where the WO2 sequence is unique. A comparisonof apo and liganded structures showed that the PFA anti-bodies change conformation by around 1 Å across the back-bone of VH CDR3 upon binding Aβ, whereas WO2 CDRsremain virtually unchanged on binding Aβ. In this respect,

Figure 1: Electrostatic surface electrostatic representation of the WO2Fab antibody (A) and the PFA1 antibody (B) incomplex with the Aβ peptide. In both representations the solid white is the heavy chain of the antibody and the translucent-dark region is the light chain. The surfaces are coloured by electrostatic potential with negative charge shown in red and,positive charge in blue. The Aβ peptide is shown as ball-and-stick and water molecules as small spheres.



WO2 represents a more rigid template for the design ofengineered proteins that recognise the immunodominant B-cell epitope of Aβ.

Figure 1 shows Aβ in the binding cleft of the PFA1 andWO2 antibodies. A point of distinction between these struc-tures is that in WO2 there is a large water filled cavity inthe heavy chain beneath Asp 7 of Aβ. Unlike PFA1, WO2makes no close contact or water mediated interaction withAsp 7 of Aβ. This may be a useful distinction between thetwo antibody binding sites considering the work of Zirahand coworkers who conducted an in vitro aging study ofaspartates in Aβ1-16 following a finding that an unusuallyhigh content of racemised and isomerised aspartates (com-mon protein damage phenomena associated with ageing)were found in Aβ from amyloid deposits (Zirah et al., 2006).Zirah and coworkers showed that the major isomer gener-ated in Aβ1-16 that was aged in vitro was L-iso-Asp 7. Asp7 is the only amino acid in the WO2 recognition epitope ofAβ not to make hydrogen bonds, salt bridges, or significantvan der Waals contacts with WO2. Rather, it appears to berigidified in the antibody by hydrogen bonds between its sidechain and that of the adjacent Arg 5 of Aβ. Taken togetherwith the fact that WO2 makes no direct contact with Asp 1of Aβ, the WO2 Fv domain represents a promising lead forthe development of an immunotherapy targeting the keyregion of Aβ, between the α- and β-secretase cleavagesites, with likely promiscuity for preserved and aged formsof the peptide.

Figure 2 shows schematic representations of the molecularbasis for PFA1 and WO2 recognition of the Aβimmunodominant B-cell epitope. In the figure, interactionsconserved between Aβ and the antibodies are highlighted inyellow, these occur predominantly at the light chain interfacewith Aβ. Again, the hydrogen bonding network that restrainsAsp 7 of Aβ in the PFA antibody is absent in the WO2mode of binding, and with Asp 7 unrestrained, theconformation of the C-terminus of this epitope is stabilisedin WO2 by hydrophobic interaction between Ser 8 of Aβand Tyr 100B (H) in CDR3 of WO2.

Despite these differences in binding, the conformation ofAβ when liganded to WO2 or PFA antibodies is remarkablysimilar across the core epitope EFRH (see superposition inFigure 3). This conformation should serve as a reliable tem-plate for the design of antigens for active immunotherapiestargeting Aβ.

Structural studies of insulin-regulated aminopeptidase

– a protein receptor involved in memory

A variety of brain functions are regulated by a disparatefamily of neuropeptides. An interesting example of these isone of the bioactive degradation products in the renin–an-giotensin system, Angiotensin IV (Ang IV; Val-Tyr-Ile-His-Pro-Phe), a metabolite that possesses very different bio-logical properties to its precursors. Intra-cranial administra-tion of Ang IV or its analogue Nle1-Ang IV has been shownto enhance memory retrieval and retention in several ani-mal models (Braszko et al., 1988; Wright et al., 1993; Wrightet al., 1999; Braszko, 2004), while its precursors Ang I toAng III have no effect. While able to facilitate learning andmemory in normal rodents, Ang IV was also able to amelio-rate the deficits in a range of rat models of amnesia, includ-ing scopolamine (Pederson et al., 1998) and mecalyminetreatment (Olson et al., 2004), ischemic damage (Wrightet al., 1996), chronic alcohol exposure (Wisniewski et al.,1993) and bilateral perforant pathway lesions (Wright etal., 1999). Ang IV is known to stimulate DNA synthesisand enhance thymidine incorporation and may be involvedin several neurobiological actions including neural develop-ment and neuron survival (Hall et al., 1995).

It has been proposed that the AT4 receptor, the only spe-cific binder of Ang IV in the brain, might be responsible forthese effects (Wright and Harding, 1994). The AT4 recep-tor was subsequently identified as the transmembrane en-zyme, insulin regulated aminopeptidase (IRAP), which oc-curs abundantly in vesicles associated with the insulin-sen-sitive glucose transporter GLUT4 in a wide variety of tis-sues (Albiston et al., 2003). Along with GLUT4, IRAP trans-locates to the plasma membrane in response to insulin andis rapidly internalised in the absence of this signal (Kanzaki,2006; Huang and Czech, 2007). IRAP belongs to the M1family of zinc metallopeptidases, which includes the ami-nopeptidases A, N and B (Rogi et al., 1996). This class ofenzymes is characterized by two common structural fea-tures - a Zn2+-binding motif HEXXH(X)

18-E and an exopep-

tidase motif GXMEN (Iturrioz et al., 2001). IRAP is a typeII membrane-spanning protein with an extracellular cata-lytic site (Keller et al., 1995; Rogi et al., 1996). The extra-cellular domain of IRAP contains 18 potential N-linkedglycosylation sites as well as a putative cleavage site (F154/A155) (Rogi et al., 1996) for release of the extracellulardomain.

Following identification of IRAP as the AT4 receptor(Albiston et al., 2003), it was shown that Ang IV was aspecific inhibitor of IRAP’s aminopeptidase activity (Lewet al., 2003). This observation has led to the hypothesis thatmodulation of IRAP’s aminopeptidase activity may be re-sponsible for the cognitive effects seen with Ang IV, possi-



Figure 2: Schematic representations of the molecular basis for PFA1 and WO2 recognition of the Aβ immunodominant B-cell epitope. This figure was produced using LIGPLOT (Wallace et al., 1995).

Figure 3: Superposition of the two Aβ 2-8 peptides when liganded to WO2 (red) and PFA (grey).



bly through prolonged circulation of neuropeptides that po-tentiate memory. This is supported by the observation thatseveral in vitro substrates of IRAP, including vasopressin(de Wied et al., 1993; Engelmann et al., 1996), CCK-8(Dauge and Lena, 1998; Fink et al., 1998; Huston et al.,1998; Sebret et al., 1999; Voits et al., 2001; Dauge et al.,2003), oxytocin (Tomizawa et al., 2003) and somatostatin(Schettini et al., 1988; DeNoble et al., 1989) have beenshown to facilitate learning and memory in rodents in aver-sion conditioning and spatial learning tasks. Testing of thishypothesis was carried out through examining the effectsof another known IRAP inhibitor, the peptide LVV-hemorphin-7, on memory and it was shown that this inhibi-tor had a very similar effect to Ang IV (Lee et al., 2004).While this does not unambiguously establish the actual

mechanism and role of the inhibitors and IRAP in improvingmemory, it adds further support to the neuropeptide protec-tion model. Other hypotheses for these effects that havebeen suggested include: (1) the regulation of GLUT4 trans-location and therefore glucose uptake and neuronal activity,or (2) the direct activation of a signalling pathway. Evidencefor the direct role of IRAP in the facilitation of this memoryenhancement remains circumstantial.

Bioinformatics

Computational analysis of the IRAP amino acid sequencesuggests that IRAP is comprised of several distinct regions(Fig. 4). Starting at the N-terminus, the cytoplasmic tail ispredicted to be primarily disordered with some α-helical

Figure 4: Diagrammatic representation of the domain arrangement of IRAP based on analysis of the IRAPamino acid sequence.

structure. Interestingly, IRAP is the only member of the M1family that has a large cytoplasmic tail and it is believed thatthis tail may be involved in the trafficking of IRAP andGLUT4. This tail is followed by a typical single pass trans-membrane region. Immediately proximal to the other sideof the membrane is a β-sheet region, followed by an α-helical region that contains the conserved active site resi-dues HEXXH(X)

18-E and GXMEN. A short β-sheet region

then separates this from the helical final region whose func-tion is unknown, although it has been proposed to act as amolecular chaperone (Rozenfeld et al., 2004).

Crystal Structures

To date no crystal structure of IRAP has been solved.Structures of several similar enzymes, however, have beendetermined including human leukotriene A4 hydrolase(LTA4H, 1HS6, 10% sequence identity to IRAP,(Thunnissen et al., 2001)), aminopeptidase N from E. coli(APN, 2DQ6, 11% sequence identity to IRAP, (Ito et al.,2006)) and N. meningitides (2GTQ, 12% sequence iden-tity to IRAP, (Nocek et al., 2008)), T. acidophilum tricornprotease interacting factor F3 (Tricorn, 1Z5H, 17% sequence

identity to IRAP, (Kyrieleis et al., 2005)) and cold aminopep-tidase from C. psychrerythraea (ColAP, 3CIA, 10% se-quence identity to IRAP, (Bauvois et al., 2008)).

LTA4H was the first M1 aminopeptidase family memberfor which a structure was solved (Fig. 5A, (Thunnissen etal., 2001)). LTA4H has a relatively short C-terminal region,similar to the more recent structure of cold aminopeptidaseand the backbones of LTA4H and ColAP superimpose wellwith a root mean square deviation (r.m.s.d.) of 1.2 Å for509 Cα atoms (Bauvois et al., 2008). They are both foldedinto three distinct domains (equivalent to D1, D2 and D4 ofthe proposed domain structure of IRAP, (Fig. 4)). D1 iscomposed of β-sheets which present a large surface to thesolvent (Fig. 5). The catalytic domain (D2) is mainly com-posed of α-helices with the active site residues located ontwo helices. The active centre is located in a groove thatconsists of one β-strand and three α-helices in what hasbeen described as a thermolysin-like catalytic domain(Thunnissen et al., 2001; Bauvois et al., 2008). D4 is α-helical and, of the three domains, has the lowest level ofsequence conservation between LTA4H and ColAP (18%identity and 1.6 Å r.m.s.d. compared to 38% and 0.8 Å for



Figure 5: Cartoon representation of the known structures from members of the M1 aminopeptidase family. Structures arecoloured by domain (using the notation in Fig. 4). (A) LTA4H. (B) ColAP. (C) E. coli APN. (D) N. meningitides APN. (E)Tricorn.



D1 and 41% and 1.2 Å for D2, respectively).

While APN (Fig. 5C and 5D) and Tricorn interactingfactor F3 (Fig: - 5E) share the three domains described forColAP and LTA4H, they contain an additional, small, bar-rel-like domain of β-strands (equivalent to D3 in the pro-posed domain structure of IRAP (Fig. 4)) located betweenthe catalytic domain, D2, and the C-terminal domain, D4.This arrangement more closely resembles the predictedsecondary structure of IRAP. As with LTA4H and ColAP,the major differences at both the sequence and 3D struc-ture levels are found in D4. One of the most striking differ-ences is in respect to the relative positioning of this domainwith respect to D1 and D2. With LTA4H, ColAP and APNthis C-terminal domain is folded back upon D2, such thatthey are situated quite close to each other. By contrast theoverall structure of the tricorn protease more closely re-sembles that of a hook, with D4 positioned further awayfrom D2, opening up the catalytic site. This means that Tri-corn protease has a deep and wide cleft with its active sitelargely exposed to solvent, although it has been proposedthat it may undergo a conformation change in D4 that maygenerate a closed-structure similar to those seen for theother M1 family members (Kyrieleis et al., 2005; Addlagattaet al., 2006). While these closed structures may only resultfrom crystal contacts the ability of crystals of E. coli APNto absorb small molecules in crystal-soaking experimentssuggests that the active site is still accessible. Movementbetween open and closed conformations may be a mecha-nism of regulating activity or substrate specificity (Ito etal., 2006).

Homology Modelling

For such large proteins with relatively low overall sequenceidentity, the crystal structures determined to date superim-pose quite well, particularly over the catalytic region. Thisprovides confidence in the reliability of homology modelsfor other members of the M1 family developed using thesestructures as templates. To date the LTA4H structure hasbeen used to generate models of the catalytic sites for APA(Rozenfeld et al., 2002; Goto et al., 2007; Claperon et al.,2008), APB (Pham et al., 2007), pyroglutamyl peptidase II(TRHDE) (Chavez-Gutierrez et al., 2006) and IRAP (Ye,et al., 2008) to try and identify key residues important in thebiological activity of these enzymes. One example wherethis has been quite spectacularly successful is in the identi-fication of residues responsible for the modulation of APAactivity by calcium (Claperon et al., 2008). Earlier studieshad identified other residues that exerted similar effects(Iturrioz et al., 2000; Goto et al., 2007), but with the modelthe authors were able to pinpoint the exact residues respon-

sible and propose a mechanism by which calcium exerts itseffects on APA. Another example of note is where modelsof TRHDE were used to try and explain the structural dif-ferences between standard M1 aminopeptidases andTRHDE, which is an omegapeptidase but still classed in theM1 family (Chavez-Gutierrez et al., 2006). By using theirmodel to identify key differences between theomegapeptidase and aminopeptidase, they were able tomutate TRHDE such that it lost its omegapeptidase activityand displayed instead alanyl-aminopeptidase activity.

The lower sequence identity over D4 reduces the reliabil-ity of models built upon this domain. Coupled with this lowerreliability is the variable orientation and close interaction ofD4 with the active site seen in the published structures,meaning that models built using the structures as templatescan only paint a limited picture of the regulation of enzymeactivity. With the availability of the new structures of M1aminopeptidases, however, more detailed models and im-proved insights will hopefully be gained.

Mechanism of activity and understanding substrate

specificity

A detailed mechanism of action for M1 aminopeptidaseactivity was proposed by Ito et al. (2006) and computationaldocking of the synthetic substrate Leu-MCA into a molecularmodel of IRAP generates highly favourable solutions showinginteractions consistent with this mechanistic description(Ye et al., 2008). Proton donation by Tyr 529 of IRAP completes hydrolysis of the N-terminal peptide bond of the substrate with interactions with the zinc ion stabilising the transition (Figure 6).

The crystal structures provide structural support for otherbiochemical data in proposing a mechanism for cleavage.These structures by themselves, however, have not pro-vided significant insight into the substrate specificity of theM1 family, particularly considering the active site regionshows high sequence and structural similarity. It is apparentthat only small changes are responsible for determining sub-strate specificity. For example, only two mutations resultedin the change of TRHDE from an omegapeptidase to anaminopeptidase (Chavez-Gutierrez et al., 2006). Models ofother members of the M1 family can used to provide in-sights into this question and to help design mutants to fur-ther understand substrate specificity.

With this aim in mind we compared our model of IRAP toLTA4H. Two key residues, Ala 427 and Leu 483, were iden-tified which presented a more open arrangement of the S1subsite of IRAP compared to LTA4H, where the corre-



Figure 6: Schematic showing the mechanism for M1 aminopeptidase activity proposed by Ito and col-leagues (2006).

sponding residues (Tyr 267 and Phe 314) are bulky aromat-ics (Ye et al., 2008). Alteration of the S1 subsite of IRAPby the corresponding mutations resulted in altered activityand substrate specificity. The IRAP mutant L483F, althoughcapable of efficiently cleaving the N-terminal Cys fromvasopressin, was unable to cleave the tyrosine residue fromeither leu-enkephalin or cyt6-vasopressin. The mutant alsoshowed a dramatic reduction in the affinity of the inhibitornorleucine1-angiotensin IV whereas the affinity of Ang IVremained unaltered. Introducing an A427Y mutation to IRAP,by contrast, resulted in increased binding affinity of the pep-tide substrates without a corresponding increase in the rateof cleavage. This not only suggests a specific role for the

two residues, Ala 427 and Leu 483, in defining the catalyticcleft of IRAP but confirms that substrate specificity is likelya result of small differences imparted from the chemicalnature of the residues between the family members.

Inhibitors

The development of new, specific M1 aminopeptidaseinhibitors is a difficult and complicated task due to the highsequence identity and structural homology of the catalyticsites of these proteins. The multiple biological roles of mem-bers of the M1 aminopeptidase family, however, requirehighly specific inhibitors for use both therapeutically and for



physiological investigations. The existing crystal structuresof family members, however, do provide a basis for attempt-ing the rational development of selective inhibitors. An ex-ample of this is the use of the structure of LTA4Hcomputationally docked to a known inhibitor to develop apharmacophore model then used to identify a subset of com-pounds that inhibited LTA4H activity (Grice et al., 2008).One of these compounds was a high affinity inhibitor invitro, orally bioavailable and showed in vivo activity.

Most physiological studies of IRAP activity have utilisedthe two known peptide inhibitors, Ang IV and LVV-H7, andtheir modified forms (Albiston et al., 2007). These havesome key limitations, including their short half-life and pro-miscuous inhibitory activity. One way of overcoming someof these limitations has been the development of cyclic pep-tide inhibitors that have higher IRAP affinity and longer half-lives (Axen et al., 2006; Axen et al., 2007). Unfortunatelythese cyclic peptides are still not selective enough for thera-peutic use or detailed physiological investigation; for examplethe best cyclic inhibitor also inhibits APN.

We have taken a slightly different approach to our at-tempts to identify new inhibitors. Using a model of the ac-tive site of IRAP based upon the crystal structure of LTA4H,we used in silico screening and in vitro assays, some ofthe latter performed in the lab of our collaborators, DrsAnthony Albiston and Siew Yeen Chai, to identify a familyof highly specific, competitive, non-peptide inhibitors(Albiston et al., 2008). Since these compounds are not pep-tidic but instead small molecule inhibitors they are amenableto medicinal chemistry modification to tune their activitiesand pharmacokinetic properties. Members of the family ofcompounds show negligible activity against some of IRAP’sclosest relatives, including ERAP1, ERAP2, APN andLTA4H. When administered intracranially they showed thesame memory enhancement effects seen for classic IRAPinhibitors Ang IV and LVV-H7, providing further supportfor the effects being mediated directly through IRAP. Theseinhibitors are providing the basis for ongoing research intothe development of a new class of therapeutics for the treat-ment of memory amelioration associated with Alzheimer’sand more generally, other dementias.

Acknowledgments

We would like to thanks our many collaborators in ourstructural biology studies of Alzheimer’s disease proteins.In particular we thank Kevin Barnham, Roberto Cappai,Colin Masters, Anthony Albiston, Siew Yeen Chai and FredMendelsohn. This work was supported by a grant from theNational Health and Medical Research Council of Austra-

lia (NHMRC). D.B.A. is an Australian Postgraduate AwardScholar and a recipient of a St Vincent’s Institute Founda-tion Scholarship sponsored by Colin North and Major Engi-neering. M.W.P. is an Australian Research Council Fed-eration Fellow and an NHMRC Honorary Fellow.

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TitleAuthorsAffiliationsCorresponding authorDatesCitationCopyright

AbstractKeywordsAbbreviationsIntroductionStructural Studies of an Amyloid Peptide-antibodyComplex

Crystal StructuresStructural Studies of Insulin-regulated Aminopeptidase– a Protein Receptor Involved in Memory

Crystal StructuresBioinformatics

Homology ModellingMechanism of Activity and Understanding SubstrateSpecificityInhibitors

AcknowledgmentsFiguresFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6

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