Promising Tools in Prostate Cancer Research:Selective Non-Steroidal Cytochrome P450 17A1 Inhibitors

Cytochrome P450 17A1 (CYP17A1) is an important target in the treatment of prostate cancer because it produces androgens required for tumour growth. The FDA has approved only one CYP17A1 inhibitor, abiraterone, which contains a steroidal scaffold similar to the endogenous CYP17A1 substrates. Abiraterone is structurally similar to the substrates of other cytochrome P450 enzymes involved in steroidogenesis, and interference can pose a liability in terms of side effects. Using non-steroidal scaffolds is expected to enable the design of compounds that interact more selectively with CYP17A1. Therefore, we combined a structure-based virtual screening approach with density functional theory (DFT) calculations to suggest non-steroidal compounds selective for CYP17A1. In vitro assays demonstrated that two such compounds selectively inhibited CYP17A1 17α-hydroxylase and 17,20-lyase activities with IC(50) values in the nanomolar range, without affinity for the major drug-metabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells

Non-steroidal CYP17A1 Inhibitors: Discovery and Assessment.

CYP17A1 is an enzyme that plays a major role in steroidogenesis and is critically involved in the biosynthesis of steroid hormones. Therefore, it remains an attractive target in several serious hormone-dependent cancer diseases, such as prostate cancer and breast cancer. The medicinal chemistry community has been committed to the discovery and development of CYP17A1 inhibitors for many years, particularly for the treatment of castration-resistant prostate cancer. The current Perspective reflects upon the discovery and evaluation of non-steroidal CYP17A1 inhibitors from a medicinal chemistry angle. Emphasis is placed on the structural aspects of the target, key learnings from the presented chemotypes, and design guidelines for future inhibitors

Deorphanizing Human Cytochrome P450 Enzymes CYP4A22 and CYP4Z1 through Mechanistic in silico Modeling

Cytochrome P450 (CYP) enzymes are monooxygenases that catalyze the oxidation of structurally diverse substrates and are present in various lifeforms, including humans. Human CYPs catalyze the metabolism of xenobiotics including drugs and are involved in the essential biosynthesis of steroids, vitamins, and lipids. CYP-catalyzed metabolism and biosynthesis has been extensively studied recently, but several CYPs remain understudied despite their potential role in key biotransformation pathways. For these so-called ‘orphaned CYPs’, physiological function and structure are yet unknown, such as for CYP4A22 and 4Z1. CYP4A22 catalyzes the ω-hydroxylation of arachidonic acid to the angiogenic 20-hydroxyeicosatetraenoic acid. CYP4Z1 is overexpressed in breast cancer and other malignancies, which is correlated with tumor progression. Hence, CYP4Z1 is considered a promising breast cancer target that was not previously addressed by small molecule inhibitors. Here, we report our efforts to deorphanize CYP4A22 and 4Z1 together with our experimental partner Prof. Bureik. We were the first to predict the structure of CYP4A22 and 4Z1 by homology modeling and overcame the challenge of low-sequence similarity templates by incorporating substrate activities. We applied substrate docking and 3D pharmacophore modeling to rationalize how the binding site structure determines structure-activity relationships (SAR) trends. The well-known structural flexibility of CYPs was partially accounted for by molecular dynamics simulations. For the first time, enzyme-substrate interactions dynamics were analyzed with our novel dynamic pharmacophore approach, which led to the prediction of key residues. For CYP4A22, a residue influencing ω-hydroxylation (Phe320) and two binding residues (Arg96 and Arg233) were predicted. For CYP4Z1, the key role of Arg487 and assisting role of Asn381 for substrate binding were predicted, which was validated by in vitro mutational studies. The thereby validated CYP4Z1 model and substrate SAR were used in a virtual screening campaign resulting in a new potent and selective CYP4Z1 inhibitor (IC50: 63 ± 19 nM). Taken together, we established an in vitro/in silico deorphanization protocol that shed light on the structure-function relationships of CYP4A22 and 4Z1. This enabled us to discover a potent inhibitor of CYP4Z1 that will allow further studies on the physiological and pathophysiological role of the enzyme and might be further improved to target CYP4Z1 in a new therapeutical approach. Similar workflows could easily be applied to study other neglected enzymes in metabolism and other biotransformation pathways.Cytochrom P450 (CYP)-Enzyme sind Monooxygenasen, die die Oxidation strukturell diverser Substrate katalysieren und in verschiedenen Lebensformen, einschließlich des Menschen, vorkommen. Menschliche CYPs katalysieren den Metabolismus von Xenobiotika einschließlich Arzneistoffen und sind an der essenziellen Biosynthese von Steroiden, Vitaminen und Lipiden beteiligt. CYP-katalysierter Metabolismus und Biosynthese wurden in der Vergangenheit intensiv untersucht, aber einige CYPs sind trotz ihrer potenziellen Rolle in wichtigen Biotransformationswegen noch wenig erforscht. Für diese so genannten „orphaned“ oder „verwaisten“ CYPs, sind physiologische Funktion und Struktur noch unbekannt, wie z.B. CYP4A22 und 4Z1. CYP4A22 katalysiert die ω-Hydroxylierung von Arachidonsäure zu der angiogenen 20-Hydroxyeicosatetraensäure. CYP4Z1 wird bei Brustkrebs und anderen malignen Erkrankungen überexprimiert, was mit der Tumorprogression korreliert ist. Daher wird CYP4Z1 als ein vielversprechendes Brustkrebs-Target angesehen, das bisher nicht durch niedermolekulare Inhibitoren adressiert wurde. Hier berichten wir über unsere Bemühungen, CYP4A22 und 4Z1 zusammen mit unserem experimentellen Partner Prof. Bureik zu deorphanisieren. Wir waren die Ersten, die die Struktur von CYP4A22 und 4Z1 durch Homologiemodellierung vorhersagten und überwanden die Herausforderung der Templates mit geringer Sequenzähnlichkeit, indem wir Substrataktivitäten mit einbezogen. Wir wendeten Substrat-Docking und 3D-Pharmakophor-Modellierung an, um zu rationalisieren, wie die Struktur der Bindungstasche die Trends der Struktur-Aktivitäts-Beziehungen (SAR) bestimmt. Die bekannte strukturelle Flexibilität von CYPs wurde partiell durch Molekulardynamik-Simulationen berücksichtigt. Zum ersten Mal wurde die Dynamik der Enzym-Substrat-Interaktionen mit unserem neuartigen dynamischen Pharmakophor-Ansatz analysiert, was zur Vorhersage von wichtigen Aminosäuren führte. Für CYP4A22 wurde eine Aminosäure, die die ω-Hydroxylierung beeinflusst (Phe320) und zwei Bindungsaminosäuren (Arg96 und Arg233) vorhergesagt. Für CYP4Z1 wurde die Schlüsselrolle von Arg487 und die unterstützende Rolle von Asn381 für die Substratbindung vorhergesagt, welche durch in vitro Mutationsstudien validiert wurde. Das dadurch validierte CYP4Z1-Modell und die Substrat-SAR wurden in einer virtuellen Screening-Kampagne verwendet, die zu einen neuen potenten und selektiven CYP4Z1-Inhibitor führte (IC50: 63 ± 19 nM). Zusammengenommen haben wir ein in vitro/in silico Deorphanisierungsprotokoll etabliert, welches die Struktur-Funktionsbeziehungen von CYP4A22 und 4Z1 beleuchtet. Dies versetzte uns in die Lage einen potenten Inhibitor von CYP4Z1 zu entdecken, der weitere Studien über die physiologische und pathophysiologische Rolle des Enzyms ermöglichen wird und möglicherweise weiter verbessert werden kann, um CYP4Z1 in einem neuen therapeutischen Ansatz zu adressieren. Ähnliche Arbeitsabläufe könnte leicht angewendet werden, um andere vernachlässigte Enzyme im Metabolismus und anderen Biotransformationswegen zu untersuchen

Antimalarial drug rescue through safety improvement: design, synthesis and evaluation of amaodiaquine analogues

Includes bibliographical references.Malaria is a major cause of morbidity and mortality globally, resulting in over 200 million cases and 650, 000 deaths in 2010 according to the 2012 WHO Malaria Report. Furthermore, malaria endemicity is associated with poor economic growth. One of the greatest challenges facing malaria chemotherapy is the emergence of Plasmodium strains resistant to all known clinically used antimalarials. This underscores the need for the development of new drugs that retain efficacy against the resistant parasites. In this study, analogue-based drug design was employed as a form of drug ‘rescue’ in the development of novel potential antimalarials. The main aim was to design and synthesize analogues of the 4-aminoquinoline drug amodiaquine with potentially improved safety and efficacy profiles using prior knowledge of the drug metabolism and pharmacokinetics (DMPK), toxicity and efficacy profile of the drug. A representative set of compounds in four different series was synthesized in which the 4-aminoquinoline ring was coupled with benzothiazole, benzimidazole, benzoxazole and pyridyl rings bearing different aliphatic amines and diamines. The chemistry involved aromatic nucleophilic substitution reactions and hydrogenation of nitro aromatic compounds. Benzothiazole and benzoxazole analogues with a tertiary protonatable nitrogen were found to possess potent antiplasmodial activity against the drug resistant W2 and K1 Plasmodium falciparum strains

The prediction of mutagenicity and pKa for pharmaceutically relevant compounds using 'quantum chemical topology' descriptors

Quantum Chemical Topology (QCT) descriptors, calculated from ab initio wave functions, have been utilised to model pKa and mutagenicity for data sets of pharmaceutically relevant compounds. The pKa of a compound is a pivotal property in both life science and chemistry since the propensity of a compound to donate or accept a proton is fundamental to understanding chemical and biological processes. The prediction of mutagenicity, specifically as determined by the Ames test, is important to aid medicinal chemists select compounds avoiding this potential pitfall in drug design. Carbocyclic and heterocyclic aromatic amines were chosen because this compounds class is synthetically very useful but also prone to positive outcomes in the battery of genotoxicity assays.The importance of pKa and genotoxic characteristics cannot be overestimated in drug design, where the multivariate optimisations of properties that influence the Absorption-Distribution-Metabolism-Excretion-Toxicity (ADMET) profiles now features very early on in the drug discovery process.Models were constructed using carboxylic acids in conjunction with the Quantum Topological Molecular Similarity (QTMS) method. The models produced Root Mean Square Error of Prediction (RMSEP) values of less than 0.5 pKa units and compared favourably to other pKa prediction methods. The ortho-substituted benzoic acids had the largest RMSEP which was significantly improved by splitting the compounds into high-correlation subsets. For these subsets, single-term equations containing one ab initio bond length were able to accurately predict pKa. The pKa prediction equations were extended to phenols and anilines.Quantitative Structure Activity Relationship (QSAR) models of acceptable quality were built based on literature data to predict the mutagenic potency (LogMP) of carbo- and heterocyclic aromatic amines using QTMS. However, these models failed to predict Ames test values for compounds screened at GSK. Contradictory internal and external data for several compounds motivated us to determine the fidelity of the Ames test for this compound class. The systematic investigation involved recrystallisation to purify compounds, analytical methods to measure the purity and finally comparative Ames testing. Unexpectedly, the Ames test results were very reproducible when 14 representative repurified molecules were tested as the freebase and the hydrochloride salt in two different solvents (water and DMSO). This work formed the basis for the analysis of Ames data at GSK and a systematic Ames testing programme for aromatic amines. So far, an unprecedentedly large list of 400 compounds has been made available to guide medicinal chemists. We constructed a model for the subset of 100 meta-/para-substituted anilines that could predict 70% of the Ames classifications. The experimental values of several of the model outliers appeared questionable after closer inspection and three of these have been retested so far. The retests lead to the reclassification of two of them and thereby to improved model accuracy of 78%. This demonstrates the power of the iterative process of model building, critical analysis of experimental data, retesting outliers and rebuilding the model.EThOS - Electronic Theses Online ServiceEPSRCGlaxoSmithKlineGBUnited Kingdo

Recent Developments in Flavin-Based Catalysis:Enzymatic Sulfoxidation

The synthesis of optically active sulfoxides, compounds due to their unique properties, has been a main target for synthetic organic chemistry. Recent efforts in the field of biocatalysis have allowed the preparation of enantiopure sulfoxides starting from the corresponding sulfides while using relatively mild conditions. In fact, several different types of redox biocatalysts have been found that can catalyze enantio- and/or regioselective sulfoxidations. The most promising group of enzymes able to perform selective sulfoxidations is the flavin-containing monooxygenases (FMOs). Enzymes containing a flavin cofactor have already been widely studied and used in organic synthesis, especially in reduction and/or oxidation processes. This chapter highlights the recent efforts in the preparation of chiral sulfoxides catalyzed by different types of flavoenzymes, with special attention to the parameters that can improve their catalytic properties. Novel approaches that allow performing selective sulfoxidations in which modified flavin systems are used are also discussed.</p