Unveiling a Novel Transient Druggable Pocket in BACE-1 through Molecular Simulations: Conformational Analysis and Binding Mode of Multisite Inhibitors

The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer's disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challenging. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments - huprine and rhein - that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8-14, 154-169 and 307-318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable 'floppy' pocket would enable the binding of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligomethylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications introduced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors

Multidimensional computational modeling of Potent BACE1 (β-Secretase) inhibitors towards Alzheimer’s disease treatment.

Doctoral Degree. University of KwaZulu-Natal, Durban.Alzheimer’s disease (AD), as a progressive multifactorial neurodegenerative abnormality of the brain, is often connected with loss or death of neurons as its primary pathogenesis. Another kind of dementia is associated with memory loss and unstable and irrational behaviors, especially among the elderly above 60 years. In South Africa, there are over four million people above the age of 60 years, with an approximation of one hundred and eighty-seven thousand living with dementia. The two distinguishing features (hallmarks) of AD are neurofibrillary tangles and β-amyloid plaques. The β-amyloid plaques result when amyloid precursor protein (APP) is cleaved by β-amyloid precursor protein cleaving enzyme1 (BACE1), otherwise known as β-secretase. Since 1999 the first BACE1 was discovered, it has become a major interest in attempting to develop drugs for the inhibition or reduction of the β-amyloid aggregates in the brain. Reducing or inhibiting the accumulation of β-amyloid has long been the target in the design of drugs for AD treatment. Having a good knowledge of the characteristic properties (BACE1) would assist in the design of potent selective BACE1 inhibitors with fewer or no side effects. Hitherto, only five drugs have been approved by the Food and Drug Administration (FDA) for the remediation of Alzheimer’s disease, and none of the approved drugs targets BACE1. In about twenty years of its discovery, several past and ongoing studies have focused on BACE1 therapeutic roles as a target in managing AD. Several attempts have previously beenmade in designing some small drugmolecules capable of good BACE1 inhibition. Some of the initially discovered BACE1 inhibitors include verubecestat, lanabecestat, atabecestat, and umibecestat (CNP-520). Although these inhibitors significantly lowered β-amyloid plaques in persons having neurological Alzheimer’s at its clinical trials (phase 3), they were suddenly terminated for some health concerns. The termination contributed to the reasons why there are insufficient BACE-targeted drugs for AD treatment. Lately, a novel potent, orally effective, and highly selective AM-6494 BACE1 inhibitor was discovered. This novel BACE1 inhibitor exhibited no fur coloration and common skin alteration, as observed with some initial BACE1 inhibitors. AM-6494 with an IC50 value of 0.4 nM in vivo is presently selected and at the preclinical phase trials. Before this study, the inhibition properties of this novel BACE1 inhibitor at the atomistic and molecular level of BACE1 inhibition remained very unclear. The first manuscript (chapter two) is a literature review on Alzheimer's disease and β-secretase inhibition: An update focusing on computer-aided inhibitor design. We provide an introductory background of the subject with a brief discussion on Alzheimer’s pathology. The review features computational methods involved in designing BACE1 inhibitors including the discontinued drugs. Using the topical keywords BACE1, inhibitor design, and computational/theoretical study in theWeb of Science and Scopus database, we retrieved over 49 relevant articles. The search years are from 2010 and 2020, with analysis conducted from May 2020 to March 2021. Our second manuscript (chapter three) reviewed BACE1 exosite-binding antibody and allosteric inhibition as an alternative therapeutic development. We studied BACE1 biological functions, the pathogenesis of the associated diseases, and the enzymatic properties of the APP site cleavage. We suggested an extensive application of advanced computational simulations in the investigation of anti-BACE1 body and allosteric exosites. It is believed that this investigation will further help in reducing the associated challenges with designing BACE1 inhibitors while exploring the opportunities in the design of allosteric antibodies. The review also revealed that some molecules exhibited dual binding sites at the active site and allosteric site. As a result, we recommend an extensive investigation of the binding free energy beyond molecular docking (such as advanced molecular dynamic simulations) as this promises to reveal the actual binding site for the compounds under investigation. Chapter four contains the detailed computational science techniques which cover the application of the vitally essential methods of molecular mechanics (MM), quantum mechanics (QM), hybrid of QM/MM, basis sets, and other computational instruments employed in this study. In the third manuscript (chapter five), we carried out computational simulations of AM-6494 and CNP- 520.CNP520 was one of the earliest BACE1 drugs that were terminated, chosen in this study forcomparative reasons. This simulation was to elucidate and understand the binding affinities of these two inhibitors at the atomistic level. We explored the quantum mechanics (QM) density functional theory (DFT) and hybrid QM/MM of Our Own N-layered Integrated molecular Orbital and Molecular Mechanics (ONIOM) in these simulations. These computational approaches helped in predicting the electronic properties of AM-6494 and CNP-520, including their binding energies when in complex with BACE1. Considering the debates on which protonated forms of Asp 32 and Asp 288 gives a more favorable binding energy, we analysed the two forms which involved the protonation and un-protonation of Asp 32 and Asp 228.The ONIOM protonated model calculation gave binding free energy of -33.463 kcal/mol (CNP-520)and 62.849 kcal/mol (AM-6494) while the binding free energy of -59.758 kcal/mol was observed for the unprotonated AM-6494 model. These results show the protonated model as a more favourable binding free energy when compared with the un-protonation AM-6494 model. Further thermochemistry processes coupled with molecular interaction plots indicate that AM-6494 has better inhibition properties thanCNP-520.However, it was observed that the protonation and the un-protonation of Asp 32 and Asp 228 modelscould adequately illustrate the interatomic binding of the ligands-BACE1 complex. To further explicate the binding mechanism, conformational and structural dynamism of AM-6494 relative to CNP-520 in complex with BACE1, we carried out advanced computational simulations in the fourth manuscript (chapter six). The extensive application of accelerated molecular dynamics simulations, as well as principal component analysis, were involved. From the results, AM-6494 further exhibited higher binding affinity with van der Waals as the predominant contributing energy relative to CNP-520. Furthermore, conformational analysis of the β-hairpin (flap) within the BACE1 active site exhibited efficient closed flap conformations in complex withAM-6494 relative to CNP-520, whichmostly alternated between closed and semi-open conformational dynamics. These observations further elucidate that AM- 6494 shows higher inhibitory potential towards BACE1. The catalytic dyad (Asp32/228), Tyr14, Leu30, Tyr71, and Gly230 constitute essential residues in both AM-6494 potencies CNP-520 at the BACE1 binding interface. The results from these extensive computational simulations and analysis undoubtedly elucidate AM-6494 higher inhibition potentials that will further help develop new molecules with improved potency and selectivity for BACE1. Besides, grasping the comprehensive molecular mechanisms of the selected inhibitors would also help in fundamental pharmacophore investigation when designing BACE1 inhibitors. Finally, the implementation of computational techniques in the designing of BACE1 inhibitors has been quite interesting. Nevertheless, the designing of potent BACE1 inhibitors through the computational application of the QM method such as the density functional theory (DFT), MM, and a hybrid QM/MM method should be extensively explored. We highly recommend that experimentalists should always collaborate with computational chemists to save time and other resources. ISIZULU ABSTRACT Iqoqa Isifo se-Alzheimer (AD), njengoba siqhubeka siyinhlanganisela yezimbangela ze- neurodegenerative engajwayelekile ebuchosheni, isikhathi esiningi kuxhumana nokulahleka noma ukufa kwama-neurons njengongqaphambili we-pathogenesis. Kungolunye uhlobo lwedementia oluhambisana nokulahlekelwa ukukhumbula kanyenokuxenga kanye nokuphanjanelwa ingqondo, ikakhulukazi kubantu abadala esebeneminyaka engaphezulu kuka-60. ENingizimu Afrikha, kunabantu abangaphezulu kwezigidi ezine abangephezulu kweminyaka ewu-60, ngokuhlawumbisela nje abayinkulungwane namashumi ayisishayangolombili nesikhombisa baphila nedemetia. Zimbili izimpawu ezihlukanisekayo ze-AD ziba-ama-neurofibrillary tangles kanye ne-B-amyloid plaques. I-B-amyloid plaques ingumphumela ngesikhathi i-amyloid eyiprotheni egijimayo iqhwakele oketshezini i-enzyme1 (BACEI), ngale kwalokho yaziwanjenge B-secretase. Kusukela ngo 1999 i-BAC1 yatholakala, isiphenduke ungqaphambili emizamweni yokwakha isidakamizwa sokwehlisa i-B-amyloid ngokwezinga lengqondo. Ngokunciphisa ukwanda kwe-B-amyloid isiphenduke okuqondiwe mayelana nokuqopha isidakamizwa ukuze kwelashwe i-AD. Ukuba nolwazi oluhle oluthinta isici sezakhi ze-BACE1 kuzosiza ekubazeni amandla akhethiwe i-BACE1 ukuvimbela imiphumela engaqondiwe. Kuze kube manje mihlanu imithi esiphasisiwe ngabezokuphatha ukudla kanye nezidakamizwa (FDA) ukwelapha isifo se-Alzheimer kanye nokuthi azikho kulezi eziphasisiwe izidakamizwa ebhekana ngqo ne-BACE1. Emva kokuba selitholakele lapho nje eminyakeni engu 20, sekunezinye esikhathini esedlule kanye nezifundo ezisaqhubeka zigxile ngokubheka kakhulu iqhaza lokwelapha i-BAC1 njengokuqondiswe ekungameleni u-AD. Imizamo eminingana yenziwa esikhathini esedlule ukuqopha uketshezi lwezidakamizwa olukwazi ukuvimba kahle i-BACE1. i-B-amyloid plaques kumuntu one-neurological ye-Alzheimer’s kumzamo (isigaba 3), kwabuye kwanqanyulwa ngenxa yokukhathazeka ngokwezempilo. Ukunqanyulwa kwanikela kuzizathu zokusilele kwezidakamizwa okuqondene nokulashwa kwe-AD. Kamuva, i-novel enamandla, ngisho ngawo umlomo kanye neyakhethwa ngezinga eliphezulu i-AM-6494 BACE1 evikelayo yatholakala. Le noveli i-BACE1 evimbayo yabukisa hhayi ukushintsha kombala woboya kanye nokushintsha kwesikhumba okujwayelekile, njengoba kubukwa nezivimbo zokuqala ze-BACE1. I-AM-6494 ne-IC50 enobumqoka buka 0.4nM kuyo i-vivo ekhethwa ngokwamanje kanye nesigaba sembulambethe yemizamo. Ngaphambi kwalesi sifundo, izakhi zesivimbela zale noveli i-BACE1zivimba ngokwe-atomistic kanye neqophelo le-molecular ye-B ACE1evimbayo kusale nje kungacacile. Umqulu wokuqala (isahluko sesibili) ukubuyekezwa kwesifo se-Alzheimer’s kanye no-B-secretase ovimbayo: ezikhumbuzayo ezigxile ngokusizwa yikhompuyutha eyisivimbo ngokwakhiwa. Sethula isendlalelo sesifundo kanye nengxoxo kafushane nezimbangela nemiphumela ye-Alzheimer. Ukubukezwa kwezimpawu zendlela zobukhompuyutha kufaka ekuqopheni isivimbo se-BACE1 nokuqhutshekiswa kwesidakamizwa. Ngokusebenzisa ofeleba begama BACE1, kusho ukwakha isivimbo, kanye nesifundo senjulalwazi kulwembu lobuchwepheshe kanye ne-Scopus sesizindalwazi. Sathola amaphepha acwaningiwe anokuhlobana angaphezulu kuka 49. Unyaka wokuthungatha usukela ku2010 kuya ku2020, nohlaziyo lwenziwa kusukela kuNhlaba 2020 kuya kuNdasa 2021. Umqulu wethu wesibili (isahluko sesithathu) sabuyekeza i-BACE ehlanganisa i-exosite antibody kanye ne-allosteric yokuthuthukisa ukwelashwa. Sakufunda ukusebenza kwesayensi yokuphila ye-BACE1, i-pathogenesis ehambisana nezifo kanye nezakhi zama-enzymatic esizinda sokuhlukana se-APP. Saphakamisa ukufakwa okunzulu nokucokeme kokulinganisa ngobuchwepheshe bekhompuyutha ekuphenyeni ama-anti-BACE1 omzimba kanye ne-allosteric ye-exosites. Kuyakholeka ukuthi uphenyo luzoqhubeka nokusiza ekwehliseni izinselelo ezihambisana nokwakha isithiyo se-BACE1 ngesikhathi kuhlolwa amathuba okwakheka kwe-allosteric yama-antibodies. Ubuyekezo luphinde lwaveza uketshezi olubukisa isizinda sokuhlanganisa kabili kusizinda esikhuthele kanye nesizinda se-allosteric. Umphumela, kube ukwenza isincomo mayelana nocwaningo olunzulu oluzohlanganisa umfutho okhululekile odlulele ku-molecular docking (njengesicokeme se-molecular yokuhlukahlukana kokulinganisa) njengoba lokhu kuthembisa ukuveza isiza esibopha ngempela ama-compounds angaphansi Isahluko sesine siqukethe imininingwane ngamaqhinga e-computational sayensi efaka isicelo esibalulekile sezindlela ezibalulekile ze-molecular mechanics (MM), i-quantum mechanics (QM), i-hybrid ye-QM/MM, ngesisekelo samasethi kanye namanye amathuluzi ekhompuyutha akhethwa kulesi sifundo. Kumqulu wesithathu (isahluko sesihlanu), siqhube isilinganiso se-computational ye-AM-6494 kanye CNP-520.I-CNP-520 kwakungenye yezidakamizwa zokuqala zeBACE1 ezashatshalaliswa, zakhethwa kulesisifundo ngezizathu zokuqhathanisa. Ukulinganisa kwakuchaza kanye nokuqonda ukusondelana ngokuhlanganiswa kwezithiyo ezimbili kusigaba se-atomistic. Kwahlolwa i-quatum mechanics (QM) yesisindo yokusebenza kwenjulalwazi (DFT) kanye ne-hybrid QM/MM yokwethu okuno-N oluwugqinsi lwe-molecular Orbital kanye ne-Molecular Mechanics (ONIOM) kulolu linganiso. Lezi zindlelakwenza ze-computational zasiza ekuqageleni kwezakhiwo zama-electronic e-AM-6494 kanye CNP-520, kungena namandla okuhlanganisa ngesikhathi kuba lukhuni ne-BACE1. Ngokucabanga izinkulumo mpikiswano mayelana nokuma kwe-protonated ye-Asp32 kanye Asp288 kunika ukuvumelana namandla okuhlanganisa, nokuhlaziya izimo ezimbili ezifaka i-protonation kanye ne-unprotonation ye-Asp32 kanye Asp228. I-ONIOM ye-protonated yomfanekiso wokubala wanikeza amandla akhululekile okuhlanganisa -33,463kcal/mol (NP-520) kanye 62.849 kcal /mol kwavela i-unprotonate ye-AM6494. Imiphumela itshengisa ukuthi i-protonated iyisifanekiso njengoba kuyisona esivumela ukuhlanganiswa ngokukhululeka ngesikhathi lapho bekuqhathanisa ne-unprotonation yomfanekiso u-AM-649. Kuqhutshelwa phambili nemisebenzi ye-thermochemistry kuhlangana nokudlelana ne-molecular plots kutshengisa ukuthi i-AM-649 inezakhiwo ezinhle zokuvimba kune CNP-520. Yize kunjalo kwabonakala ukuthi i-protonation kanye ne-unprotonation ye-Asp32 kanye neyomfanekiso owu- Asp228 bekungatshengisa ngokwenele ukuhlanganisa ngokwe-interatomic yama-ligands EBACE1 ebilukhuni. be-BACE1 ngokwedlulele isilinganiso se-computational. Ukwenza ngokujulile kuphangiswa isilinganiso se-molecular ngokuhlukana, kwakakwa nohlaziyo olusemqoka lwezingxenyana. Imiphumela ye-AM-6494 yaqhubeka yatshengisa ukusondelana kokuhlanganiswayo no-van der Waals njengohamba phambili ekunikeleni amandla ahlobene ne-CNP-520. Ukuvuma kohlaziyo lwe-B-hairpin ngaphakathi ku-BACE1 kutshengiswa esizeni esiphilayo esivala ngendlela umnyakazo wokuvuma kobunkimbinkimbi be-AM-6494 ehlobene neCNP-520, ngokuvamile eshitshashintshayo phakathi kwevalekile kanye nezishaya sakuvuleka kokuvuma okunhlobonhlobo. Lokhu kuhlolwa kuqhubeke kwachazwa ngokuthi i-AM-6494 itshengisa ukuvimba okukhulu nokunethemba mayelana ne-BACE1. Isikhuthazizinguquko se-dyad (Asp32/228), Tyr14, Leu 30, Tyr 71, kanye ne-Gly230 kwakha izinsalela ezibalulekile nxazombili kuAM-6494ne-potencies yeCNP-520 kuBACE1 nesixhumanisi esihlanganisayo. Imiphumela ivela kulama-computational anzulu ayisilinganiso kanye nohlaziyo olucacisa ngokungangabazi i-AM-6494 enesivimbelo esiphakeme esingakwazi ukuqhubeka nokusiza intuthuko yama-molecules amasha anamandla athuthukile kanye nakhethelwe i-BACE1. Ngaphandle kwalokhu, ukucosha izinkambiso ezibanzi ze-moleculor mayelana nezivimbo ezikhethiwe kuzosiza mayelana nophenyo olubalulekile lwe- pharmacophore ngesikhathi kuqoshwa izivimbo se-BACE1. Ekugcineni, ukwenziwa kwe-computational ngokwamacebo ekubazeni izivimbo ze-BACE1 kube into ehlaba umxhwele. Nokho ukubaza izivimbo ezinamandla ze-BACE1 ngokusebenzisa i-computational yendlela ye-QM njengenjulalwazi yesisindo esisebenzayo (DFT), MM, kanye nendlela ye-hybrid QM/MM kufanele iphenywe kanzulu. Sincoma kakhulu ukuthi ongoti abenza izibonisi kufanele njalo bahlangane nama-computational chemists ukonga isikhathi kanye nezinye izinsiza

Identification of vascular endothelial growth factor receptor 3 (VEGFR3) as an in vitro and in vivo substrate of the Alzheimer's Disease linked protease BACE2

The protease ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) is a key drug target in Alzheimer’s disease (AD). It catalyzes the first step in the generation of the pathogenic amyloid ß (Aß) peptide and its inhibition is therefore a promising approach to prevent or delay the onset of AD. To date however, most inhibitory compounds do not discriminate between BACE1 and its close non-amyloidogenic homologue BACE2 and therefore may lead to undesired off target effects, resulting from BACE2 biology. Therefore, future compounds require a higher selectivity for BACE1 and a biomarker is required to confirm unimpaired in vivo BACE2 activity. To replace a long lasting depigmentation assay, which is the current standard for in vivo BACE2 activity monitoring, the blood plasma of BACE2 knockout mice (B2KO) was screened and the tyrosine kinase receptor Vascular Endothelial Growth Factor 3 (VEGFR3) was identified as a putative BACE2 substrate. Subsequently, VEGFR3 was thoroughly validated as an in vitro and in vivo BACE2 substrate and the BACE2 cleavage site was determined. In direct comparison to the pigmentation readout, plasma VEGFR3 performed superior and displayed higher sensitivity and lower variance. Importantly, reduction of VEGFR3 was also detectable in the plasma of BACE inhibitor treated non-human primates (NHP) and clinical trial participants, highlighting potential for applicability in the clinical context. To test whether BACE2 cleavage may be a novel mechanism to control VEGFR3 function, downstream events of VEGFR3 signaling were monitored in primary lymphatic endothelial cells (LECs). Impairment of BACE2 dependent VEGFR3 processing was accompanied by increased activation of the VEGFR3 dependent pathways AKT and ERK and resulted into enhanced transcription of the VEGFR3 inducible genes (FOXC2) and Delta-like 4 (DLL4). As a consequence, alterations in the morphological structure and drainage efficiency of lymphatic vessels and cannot be excluded in the periphery and central nervous system (CNS). Future developments in the BACE inhibitor field need to consider these implications and plasma VEGFR3 levels may be used to control for possible of target effects from BACE2 inhibition

Construcción QSAR de redes complejas de compuestos de interés en Química Farmacéutica, Microbiología y Parasitología

El diseño para la búsqueda y desarrollo de fármacos eficaces para el tratamiento de estas enfermedades, que supriman la eliminación o la degeneración celular respectivamente, es una de las líneas de investigación más importantes dentro de la química farmacéutica. En esto entra el diseño de fármacos; el diseño de fármacos está dedicado al desarrollo de modelos matemáticos para predecir propiedades de interés para una gran variedad de sistemas químicos incluyendo moléculas de bajo peso molecular, polímeros, biopolímeros, sistemas heterogéneos, formulaciones farmacéuticas, conglomerados de moléculas e iones, materiales, nano-estructuras y otros. Este tipo de predicciones no pretenden sustituir las técnicas experimentales sino complementar las mismas ayudando a obtener nuevas moléculas activas con mayor probabilidad de éxito, con la ventaja que ello supone en términos de ahorro de tiempo, recursos materiales, y muy importante: el refinamiento y reducción en el uso de animales de laboratorio. Esta metodología se basa en el uso de cálculos por ordenador y en las nuevas tecnologías de la informática. Las cuales pueden ser usadas: Para moléculas pequeñas: a) Estudios de relación cuantitativa estructura molecular-actividad farmacológica (QSAR) y de estructura molecular propiedades toxicológicas y eco-toxicológicas incluyendo mutagenicidad e carcinogénesis (QSTR). b) Predicción de propiedades químicas y fisicoquímicas de moléculas. Estudios de relación estructura molecular y propiedades de absorción, distribución, metabolismo y eliminación (ADME). c) Predicción de mecanismos de acción biológica de moléculas y evaluación in sílico de alta eficacia para grandes bases de datos (virtual HTS). Para macromoléculas: a) Estudios de interacción fármaco-receptor (neuronas). b) Bioinformática aplicada a estudios de relación secuencia-función y propiedades estructurales de ácidos nucleicos y proteínas. c) Búsqueda de nuevas dianas terapéuticas y “sitio activo” a partir de datos de Genómica, Proteómica. d) Búsqueda de biomarcadores para diagnóstico de enfermedades o como indicadores de contaminaciones. e) Predicción de propiedades fisicoquímicas de polímeros sintéticos, biopolímeros, materiales y nano-estructuras. f) Predicción, diseño, y optimización de enzimas mutadas para procesos biotecnológicos

Initial Aβ seeds as therapeutic target for Alzheimer’s disease

Alzheimer’s disease (AD) is the leading form of dementia interfering with daily life due to progressive memory loss and cognitive disabilities. With more than 45 million people suffering from dementia worldwide, AD is one of the costliest health conditions to society. Because of an increasing proportion of elderly people the number of individuals living with dementia is expected to more than triple by 2050. Although there are symptomatic treatments available that temporarily slow the worsening of clinical symptoms, a disease-modifying cure is still missing. Therefore, dementia, and AD in particular, is becoming a public health priority evoking worldwide efforts to delay or even prevent the disease from ever developing. The amyloid cascade hypothesis explained in detail in chapter one of this thesis proposes that an impaired homeostasis of production and clearance of the amyloid-β (Aβ) peptide is the trigger initiating a sequence of pathogenic events causing AD. Aβ misfolding and aggregation leads to the accumulation of cerebral amyloid plaques, a typical hallmark of AD. As the pathology progresses, plaques continue to develop and grow, which is suggested to lead to the second disease characteristic, neurofibrillary tangles (NFTs), consisting of hyperphosphorylated tau proteins. Aβ as the driving force of this pathological cascade has been regarded as the most reasonable therapeutic target. Therefore the second chapter is dedicated to different therapeutic approaches for AD with particular focus on the β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which is responsible for regulating the production of Aβ. To this end APP transgenic (tg) mice that mimic β-amyloidosis-related features of AD were treated with a potent BACE inhibitor for six months. In response to this long-term therapy, brain Aβ as well as plaque deposition were reduced to levels comparable to six months younger animals. Surprisingly, BACE inhibition also exhibited downstream effects preventing the pathology-dependent increase of tau in the cerebrospinal fluid (CSF). Thus, BACE inhibitors are valuable therapeutic agents and their effectiveness can be predicted by CSF tau measurements in clinical trials. These findings have been published in Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association (Schelle et al., 2017, Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association, 13(6), pp.701-709). The third part of this thesis investigated the stability of small Aβ aggregates (Aβ seeds), which share pathogenic properties with the prion protein implicated in transmissible spongiform encephalopathies. Whereas in Aβ seeds inoculated APP tg mice the presence of host-derived Aβ together with the exogenously applied seeds induced their propagation, the injected material was undetectable in App null mice 30 days post inoculation. However, reinoculation with brain extracts from App null mice inoculated with Aβ seeds up to six months harboring Aβ levels below detection, still induced cerebral β-amyloidosis in APP tg hosts. In conclusion, Aβ seeds can persist even in the absence of host Aβ and regain their pathogenic activity as soon as sufficient Aβ becomes available. This discovery indicates on the one hand that lowering Aβ production inhibits the formation of new Aβ seeds and on the other hand that therapeutic intervention is most effective when applied at early stages. These results have been published in Nature Neuroscience (Ye, Fritschi, Schelle et al., 2015, Nature neuroscience, 18(11), pp.1559-61). Recent biomarker studies in familial AD subjects, which revealed that first pathological changes occur up to 25 years before clinical disease onset, supported this idea. Therefore, in the last part, pre-depositing APP tg mice were treated with a combinational therapy based on anti-Aβ immunization to remove Aβ seeds and a BACE inhibitor to block the production of soluble Aβ. Using two different APP tg mouse models results revealed that brain Aβ levels and plaque formation were reduced acutely after the treatment. Moreover, this short but early intervention delayed amyloid pathology after discontinuation of the treatment. Thus, targeting initial Aβ seeds by anti-Aβ combination therapy might be the most promising strategy to effectively prevent cerebral β-amyloidosis. These findings are now prepared for publication (Schelle et al., 2017). In summary, this doctoral thesis highlights the importance of early therapeutic intervention with anti-Aβ drugs in order to prevent AD. Previous studies have shown that treatment of AD patients who have already developed irreversible neurodegeneration might be inadequate to stop the progression of this devastating disorder and could be one reason to explain recent failures of anti-amyloid agents in numerous clinical trials. The results presented in this doctoral thesis indicate that the treatment focus should be shifted toward earlier stages of AD and even toward primary prevention before symptom onset targeting initial Aβ seeds

Structure-based Development of Secondary Amines as Aspartic Protease Inhibitors

As novel promising scaffold for HIV protease inhibition pyrrolidine-derived inhibitors have recently been reported. In this thesis the stepwise improvement of this compound class to potent inhibitors of wildtype as well as selected mutant proteases utilizing rational drug discovery methods is reported. Based on the crystal structure of a (rac)-3,4-dimethyleneamino-pyrrolidine in complex with HIV-1 protease symmetric pyrrolidine-diesters possessing the same stereochemistry were synthesized following a chiral-pool approach. The most potent compounds of the series achieve one-digit micromolar inhibition towards wild type as well as two mutant proteases (Ile50Val and Ile84Val). The cocrystal structure of one derivative in complex with the Ile84Val HIV protease revealed that two inhibitor molecules are bound in the large active site cavity comprising an area encompassed by the catalytic dyad and the flaps in the open conformation. This is the first HIV protease cocrystal structure in which the open-flap conformation of the enzyme is stabilized by an inhibitor that concomitantly addresses the catalytic dyad. As an alternative approach towards HIV protease inhibitors, the development of symmetric 3,4-bis N-alkyl sulfonamide-pyrrolidines is described. The initial lead structure possessing benzene sulfonamide groups and benzyl substituents exhibited a Ki of 2.2 µM. The X-ray structure in complex with the HIV protease enabled the rational design of a second series of inhibitors and revealed three promising symmetric substitution patterns for further lead optimization: (A) Elongation of the P1/P1’-benzyl moieties with hydrophobic substituents in para-position, (B) ortho-substitution at the P2/P2’-phenyl ring systems, and (C) para-substitution at the P2/P2’-phenyl moieties. All three strategies were pursued and resulted in inhibitors with improved affinities up to 260 nM. To elucidate the underlying factors accounting for the SAR, the crystal structures of four representatives, at least one of each modification type, in complex with HIV protease were determined. These structures provided deeper insights into the protein–ligand interactions and the underlying principles of the SAR thus enabling to choose the most promising combination of substituents in the next design cycle. The combination of these substituents rendered a final inhibitor showing a significantly improved affinity of Ki = 74 nM and the cocrystal structure in complex with the HIV protease confirmed the successful application of the pursued optimization strategy. Subsequently the influence of the active site mutations Ile50Val and Ile84Val on these inhibitors is investigated by structural and kinetic analysis. Whereas the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the crucial Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases the structural basis of this phenomenon was elucidated. Inhibitors bearing smaller N-alkyl substituents revealed a selectivity profile not being explicable with the initial SAR. By cocrystallization of the most potent derivative of a small series with HIV-1 protease, astonishingly two different crystal forms, P2(1)2(1)2(1) and P6(1)22, were obtained. Structural analysis revealed two completely different binding modes, the interaction of the pyrrolidine nitrogen atom to the catalytic aspartates being the only similarity. Encouraged by the successful utilization of cyclic secondary amines as anchoring group in the development of HIV protease inhibitors, this strategy was expanded into a general approach for lead structure identification for aspartic proteases. An initial library comprising eleven inhibitors based on easily accessible achiral linear oligoamines was developed and screened against six selected aspartic proteases (HIV-1 protease, plasmepsin II, plasmepsin IV, renin, BACE-1, and pepsin). Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was consecutively confirmed by determination of the crystal structure of two derivatives in complex with HIV-1 protease. The binding modes exhibit high similarity to the binding orientation of substrates as well as to that of peptidomimetic inhibitors. Using this information, a generalization of this binding situation to other aspartic proteases appears reasonable, thus providing a first insight into the observed structure-activity relationships

Chronic BACE-1 Inhibitor Administration in TASTPM Mice (APP KM670/671NL and PSEN1 M146V Mutation): An EEG Study

Objective: In this exploratory study, we tested whether electroencephalographic (EEG) rhythms may reflect the effects of a chronic administration (4 weeks) of an anti-amyloid β-site amyloid precursor protein (APP) cleaving enzyme 1 inhibitor (BACE-1; ER-901356; Eisai Co., Ltd., Tokyo, Japan) in TASTPM (double mutation in APP KM670/671NL and PSEN1 M146V) producing Alzheimer's disease (AD) amyloid neuropathology as compared to wild type (WT) mice. Methods: Ongoing EEG rhythms were recorded from a bipolar frontoparietal and two monopolar frontomedial (prelimbic) and hippocampal channels in 11 WT Vehicle, 10 WT BACE-1, 10 TASTPM Vehicle, and 11 TASTPM BACE-1 mice (males; aged 8/9 months old at the beginning of treatment). Normalized EEG power (density) was compared between the first day (Day 0) and after 4 weeks (Week 4) of the BACE-1 inhibitor (10 mg/Kg) or vehicle administration in the 4 mouse groups. Frequency and magnitude of individual EEG delta and theta frequency peaks (IDF and ITF) were considered during animal conditions of behaviorally passive and active wakefulness. Cognitive status was not tested. Results: Compared with the WT group, the TASTPM group generally showed a significantly lower reactivity in frontoparietal ITF power during the active over the passive condition (p < 0.05). Notably, there was no other statistically significant effect (e.g., additional electrodes, recording time, and BACE-1 inhibitor). Conclusions: The above EEG biomarkers reflected differences between the WT and TASTPM groups, but no BACE-1 inhibitor effect. The results suggest an enhanced experimental design with the use of younger mice, longer drug administrations, an effective control drug, and neuropathological amyloid markers

From Active-Site Mapping to Lead Discovery using Fragment-based Approaches on the Aspartic protease Endothiapepsin

The work focuses on the evaluation and comparison of different fragment-based approaches, for which purpose the model system Endothiapepsin (EP) has been used. The enzyme belongs to the family of aspartic proteases. We used the 361-entry in-house fragment library compiled with physico-chemical properties similar to the rule-of-three. We applied six different techniques to screen the fragment library: saturation-transfer difference NMR (STD-NMR), thermal shift assay (TSA), reporter-displacement assay (RDA), microscale thermophoresis (MST), fluorescence-based biochemical assay (HCS), and native mass spectrometry (MS). Alarmingly, the overlap between the hits produced by all six methods was very low. While only 41 out of all 361 fragment library entries could be identified by at least two methods, only 3 were identified by five methods, and no single fragment was identified as hit by all six methods taken together (chapter 2). We thus performed X-ray crystallography with individual fragments soaked to beforehand prepared apo EP crystals. For this purpose very high fragment concentrations of 90 mM were used. Intriguingly, we were able to identify 71 fragment hits as bound to EP, corresponding to 20% hit rate. Worryingly, only 30% of the 71 hits were predicted by only one of the beforehand applied screening techniques, clearly emphasizing that any screening strategy comprising at least two prescreening methods would have been able to identify only 19 (27%) of the 71 crystallographic hits. We divided the hits into two main groups: catalytic dyad binders and remote binders. Chapter 3 deals with the catalytic dyad binders. Therein we describe a variety of warheads which address the catalytic aspartates either directly or via the catalytic water molecule W501, while occupying the S1 and S1’ binding pockets. Furthermore, we found fragments, which occupied both pockets simultaneously, offering an optimal platform for further optimization strategies into both directions. The very useful information regarding preferred spots of binding remote from the catalytic dyad is described in detail in chapter 4. The so called hot spots represent key pocket residues, which have to be addressed in an optimization procedure in order to achieve optimal ligand binding. The high-throughput potential of X-ray crystallography (chapters 3 and 4) compared to the other six prescreening techniques presented in chapter 2 is still very low. Because of this, a lot of effort has been invested in academia and industry to extend the method’s applicability as a primary screening technique. Whenever used as such, practitioners exposed protein crystals to a mixture of compounds to accelerate the hit identification. However, we cannot recommend the use of cocktail experiments with clear conscience as we believe that parameters such as compound solubility, chemical reactivity, and crystal damage, often resulting in reduced ligand occupancy, diffuse electron density, or deviating fragment binding poses are provoked by the presence of multiple compounds in a mixture. These issues are exemplified in chapter 5 where we directly compared the difference electron density in the binding pocket of EP between singly soaked fragments and cocktails prepared those. For example, many of the fragments used in a mixture of two failed to be identified as hits, whereas clear density around those fragments could be observed in the single soaking experiments. Moreover, the clear assignment of a fragment to diffuse electron density usually requires additional experiments, which confirm the binding site. When considering this, even more time has to be spent, which extremely slows down the hit identification procedure. The only plausible reason to use several compounds in a mixture is when the reaction between those is aimed. This is an approach named dynamic combinatorial chemistry (DCC) and described in chapter 6. The compounds, hydrazides and aldehydes, chosen for the mixtures react with one another with dehydration to larger acylhydrazones. The formation of the strongest binders among others, acylhydrazones (S)-H4-A4 and (R)-H3-A5, was induced by the natural selection by the target protein EP. Moreover, in a follow-up optimization project, which started based on the binding modes of the two acylhydrazones, the combination of a bis-aldehyde with hydrazides resulted in the natural selection of a bis-acylhydrazone by EP, with an 240-fold improved potency compared to the initial acylhydrazones