Design of inhibitors of cathepsin K and autolysin with application of chemometrics and structure-based drug design

V raziskavah v sklopu doktorske disertacije smo se posvetili načrtovanju in eksperimentalnemu vrednotenju zaviralcev katepsina K (Cat K) in avtolizina E (AtlE). V prvem sklopu doktorskega dela smo preverili hipotezo, da s pomočjo opisa kemijske strukture zaviralcev Cat K v kompleksu ligand-encim dobimo mehanistično sliko delovanja učinkovine in s tem omogočimo optimizacijo zaviralcev z namenom zmanjševanja neželenih stranskih učinkov. Cat K je lizosomska proteaza, ki igra pomembno vlogo pri nekaterih boleznih, kot so osteoporoza, debelost, diabetes, Pagetova bolezen in ateroskleroza. V zadnjem obdobju so bile raziskave v farmacevtski industriji usmerjene predvsem v razvoj kovalentnih reverzibilnih zaviralcev Cat K za zdravljenje osteoporoze, predvsem tistih z nitrilno oziroma ketonsko funkcionalno skupino. Več zaviralcev Cat K, vključno z balikatibom, je bilo testiranih v kliničnih študijah. Balikatib se je izkazal kot učinkovit zaviralec, vendar so stranski učinki, povezani z lizosomotropizmom, ki je posledica bazičnega značaja balikatiba, onemogočili nadaljnja klinična testiranja. Osnovna spojina vodnica, ki smo jo optimizirali, je bila balikatib, pri kateri so v kliničnih študijah opazili neželene stranske učinke zaradi prevelike bazičnosti substituent na osnovnem skeletu. Najprej smo zgradili validiran kvantitativni model odnosa med strukturo in delovanjem (QSAR) za aminonitrile z benzamidno skupino. Da bi pojasnili izbor strukturnih deskriptorjev, smo uvedli primerjalni indeks prekrivanja/simetrije odzivne površine z izbranimi nivoji v vhodni plasti umetne nevronske mreže. Ugotovili smo, da so pomembni tako deskriptorji, ki so povezani s kovalentnimi vezavnimi parametri v S1 vezavnem žepu, kot tudi deskriptorji, ki opisujejo 3D strukturo molekul in njihovih nekovalentnih interakcij v kompleksu zaviralec-encim v S2 in S3 vezavnih mestih Cat K. V nadaljnjih raziskavah smo z uporabo virtualne kombinatorične knjižnice, QSAR modeliranja in molekulskega sidranja razvili novo serijo zaviralcev Cat K, ki temelji na N-(funkcionaliziranem benzoil)-homociklolevcil-glicinonitrilnem skeletu. Da bi se izognili težavam poznanih zaviralcev Cat K, povezanih z lizosomotropizmom bazičnih spojin in posledično neželenimi učinki, smo na pozicijo P3 vključili šibko bazične oziroma nebazični strukturni fragment. Tri sintetizirane spojine so bile visoko selektivne za Cat K v primerjavi s katepsini L in S z vrednostmi konstante inhibicije (Ki) v območju 10-30 nM. Študije encimske kinetike so pokazale, da imajo novi zaviralci tesen in reverzibilen način vezave na Cat K, medtem ko so kristalne strukture encima v kompleksu z zaviralcem razkrile tako kovalenten kot tudi nekovalenten način vezave med nitrilno skupino zaviralcev in katalitičnim cisteinom Cys25. V drugem delu raziskav smo se posvetili iskanju novih potencialnih zaviralcev AtlE iz bakterijskga seva Staphylococcus aureus (S. aureus). Peptidoglikan, poznan tudi kot murein, je glavna sestavina bakterijske celične stene in je v nenehnem ravnovesnem procesu sinteze in razgradnje, kar je temelj za normalno delovanje bakterijskih celic. Avtolizine uvrščamo v skupino peptidoglikanskih hidrolazto so encimi, ki igrajo ključno vlogo pri razgradnji bakterijske celične stene. Med procesom celične delitve so avtolizini odgovorni za ločevanje ekvatorialnega septuma med nastajajočima hčerinskima bakterijskima celicama. Pri mutantih, ki so imeli izbit gen za avtolizine, so se pojavile resne motnje celične delitve, nastale so velike gruče celic, ki niso tvorile biofilma. Bakterijski avtolizini kot potencialne tarče tako predstavljajo še dokaj novo, a obetavno področje raziskav protimikrobnih učinkovin. Kot izhodišče pri načrtovanju potencialnih zaviralcev AtlE smo uporabili še neobjavljeno kristalno strukturo AtlE z vezanim ogljikohidratnim ligandom. Z računalniškimi metodami strukturno podprtega načrtovanja in farmakofornimi modeli smo uspeli dobiti serijo potencialnih zaviralcev, ki imajo afiniteto vezave v nizkem mikromolarnem območju. Pri tem smo uporabili tri glavne pristope racionalnega načrtovanja novih potencialnih zaviralcev AtlE: (1) načrtovanje na osnovi mimetikov sladkorja v kristalni strukturi, (2) načrtovanje na osnovi mimetikov prehodnega stanja glede na teoretični mehanizem katalize substrata in (3) načrtovanje na osnovi zadetkov iz virtualnega rešetanja glede na dostopno kristalno strukturo AtlE. Afiniteto vezave med AtlE in izbranimi spojinami smo določili s površinsko plazmonsko resonanco (SPR) in mikrotermoforezo (MST). Identificirali smo benzamido-piperidinil-ureido-fenilni kemijski razred spojin z afiniteto vezave do AtlE v nizkem mikromolarnem območju. Najbolj obetavne potencialne zaviralce bomo obravnavali v nadaljnjih študijah kristalizacije z AtlE in s celičnimi testi tvorbe biofilma.Our studies in the framework of the doctoral dissertation were devoted to the design and experimental evaluation of the inhibitors of cathepsin K (Cat K) and autolysin E (AtlE). In the first part of our work we have tested the hypothesis that using the information, obtained from chemical structures of Cat K inhibitors, could yield mechanistic picture of the inhibitor´s mechanism of action and thereby allowing subsequent known inhibitors optimization with the main goal of reducing their side effects. Cat K is a lysosomal protease, which plays an important role in various diseases, such as osteoporosis, obesity, diabetes, Paget\u27s disease and atherosclerosis. In recent years, focus of the pharmaceutical industry has been directed towards development of covalent, reversible inhibitors of Cat K for the treatment of osteoporosis, particularly inhibitors with a nitrile or ketone warheads. Several Cat K inhibitors, including balicatib, have been tested in clinical studies. Balicatib proved to be an effective inhibitor, however the side effects associated with lysosomotrophism as a result of a basic character of the balicatib, prevented further clinical trials. We have optimized lead compound balicatib, which in clinical studies showed undesirable side effects due to its excessive basicity of the substituents attached to its main scaffold. Firstly, we have built a validated quantitative structure-activity relationships model (QSAR) for benzamide-containing aminonitriles. In order to elucidate the selection of the structural descriptors, we have introduced an index of overlap/response surface symmetry of the output layer and the selected levels in the input layer of the artificial neural network. Descriptors, found to be important, are linked by covalent binding parameters in the S1 Cat K binding pocket, as well as descriptors that describe the 3D structure of the molecules and their non-covalent interaction in S2 and S3 binding sites of the inhibitor:enzyme complex. In our further studies, we designed a new series of Cat K inhibitors based on N-(functionalized benzoyl)-homocycloleucyl-glycinonitrile scaffold by using virtual combinatorial chemistry, QSAR modeling, and molecular docking studies. In order to avoid issues of known Cat K inhibitors v associated with lysosomotrophism of the basic compounds which can lead to several side effects, we have introduced a weakly basic or non-basic structural fragment on the P3 position. Three synthesized compounds were highly selective for Cat K when compared with cathepsins L and S with the Ki values in the 10-30 nanomolar range. The kinetic studies revealed that the new compounds exhibited reversible tight binding to Cat K, while the X-ray structural studies showed covalent and non-covalent binding between the nitrile group and the catalytic cysteine Cys25 site. The second part of our research was dedicated to development of novel potential AtlE inhibitors of bacterial strain Staphylococcus aureus (S. aureus). Peptidoglycan, also known as murein, is the main component of the bacterial cell wall and is in constant process of synthesis and degradation, which is crucial for the normal functioning of the bacterial cells. Autolysins belong to the enzyme family of peptidoglycan hydrolasesthese are enzymes that play a key role in the degradation of the bacterial cell wall. During the process of cell division, autolysins are responsible for the separation of the equatorial septum of the forming daughter cells. In the mutants with AtlE knocked out gene, serious disturbance of the cell division have occurred, resulting in the large, biofilm-negative clusters. Therefore, bacterial autolysins represent a potential promising new drug target for the development of the antibacterial agents. We used unpublished crystal structure of AtlE in a complex with carbohydrate ligand as a starting point for the development of novel AtlE inhibitors. Using computer-aided structural drug design methods and pharmacophore modeling, we managed to obtain a novel series of potential inhibitors, which had binding affinities in the low micromolar range. We employed three main approaches of the rational drug design for new potential inhibitors of the AtlE: (1) strategy based on crystalized sugar ligand mimetics, (2) strategy based on mimetics of transition state in connection with theoretical mechanism of substrate binding and catalysis, and (3) strategy based on the results from the large scale virtual screening campaign utilizing the available AtlE crystal structure. The binding affinity between the AtlE and potential inhibitors was measured by Surface Plasmon Resonance (SPR) and microscale thermophoresis (MST). We identified benzamido-piperidinyl-ureido-phenyl chemical class of compounds with a binding affinities to AtlE in the low micromolar range. The most promising potential inhibitors will be subjected to x-ray crystallization studies and cell biofilm formation assays