Cytotoxic Activity of α-Aminophosphonic Derivatives Coming from the Tandem Kabachnik-Fields Reaction and Acylation.

Encouraged by the significant cytotoxic activity of simple α-aminophosphonates, a molecular library comprising phosphonoylmethyl- and phosphinoylmethyl-α-aminophosphonates, a tris derivative, and N-acylated species was established. The promising aminophosphonate derivatives were subjected to a comparative structure-activity analysis. We evaluated 12 new aminophosphonate derivatives on tumor cell cultures of different tissue origins (skin, lung, breast, and prostate). Several derivatives showed pronounced, even selective cytostatic effects. According to IC50 values, phosphinoylmethyl-aminophosphonate derivative 2e elicited a significant cytostatic effect on breast adenocarcinoma cells, but it was even more effective against prostatic carcinoma cells. Based on our data, these new compounds exhibited promising antitumor activity on different tumor types, and they might represent a new group of alternative chemotherapeutic agents.

Dual Inhibitors of AChE and BACE-1 for Reducing Aß in Alzheimer's Disease: From In Silico to In Vivo.

Alzheimer's disease (AD) is a complex and widespread condition, still not fully understood and with no cure yet. Amyloid beta (Aß) peptide is suspected to be a major cause of AD, and therefore, simultaneously blocking its formation and aggregation by inhibition of the enzymes BACE-1 (ß-secretase) and AChE (acetylcholinesterase) by a single inhibitor may be an effective therapeutic approach, as compared to blocking one of these targets or by combining two drugs, one for each of these targets. We used our ISE algorithm to model each of the AChE peripheral site inhibitors and BACE-1 inhibitors, on the basis of published data, and constructed classification models for each. Subsequently, we screened large molecular databases with both models. Top scored molecules were docked into AChE and BACE-1 crystal structures, and 36 Molecules with the best weighted scores (based on ISE indexes and docking results) were sent for inhibition studies on the two enzymes. Two of them inhibited both AChE (IC50 between 4-7 µM) and BACE-1 (IC50 between 50-65 µM). Two additional molecules inhibited only AChE, and another two molecules inhibited only BACE-1. Preliminary testing of inhibition by F681-0222 (molecule 2) on APPswe/PS1dE9 transgenic mice shows a reduction in brain tissue of soluble Aß42.

Structure-Activity Relationships of 8-Hydroxyquinoline-Derived Mannich Bases with Tertiary Amines Targeting Multidrug-Resistant Cancer.

A recently proposed strategy to overcome multidrug resistance (MDR) in cancer is to target the collateral sensitivity of otherwise resistant cells. We designed a library of 120 compounds to explore the chemical space around previously identified 8-hydroxyquinoline-derived Mannich bases with robust MDR-selective toxicity. We included compounds to study the effect of halogen and alkoxymethyl substitutions in R5 in combination with different Mannich bases in R7, a shift of the Mannich base from R7 to R5, as well as the introduction of an aromatic moiety. Cytotoxicity tests performed on a panel of parental and MDR cells highlight a strong influence of experimentally determined pKa values of the donor atom moieties, indicating that protonation and metal chelation are important factors modulating the MDR-selective anticancer activity of the studied compounds. Our results identify structural requirements increasing MDR-selective anticancer activity, providing guidelines for the development of more effective anticancer chelators targeting MDR cancer.

Rapid Identification of Potential Drug Candidates from Multi-Million Compounds' Repositories. Combination of 2D Similarity Search with 3D Ligand/Structure Based Methods and In Vitro Screening.

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost-effective approach in early drug discovery. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compounds' databases. This approach can be combined with physico-chemical parameter and diversity filtering, bioisosteric replacements, and fragment-based approaches for performing a first round biological screening. Our objectives were to investigate the combination of 2D similarity search with various 3D ligand and structure-based methods for hit expansion and validation, in order to increase the hit rate and novelty. In the present account, six case studies are described and the efficiency of mixing is evaluated. While sequentially combined 2D/3D similarity approach increases the hit rate significantly, sequential combination of 2D similarity with pharmacophore model or 3D docking enriched the resulting focused library with novel chemotypes. Parallel integrated approaches allowed the comparison of the various 2D and 3D methods and revealed that 2D similarity-based and 3D ligand and structure-based techniques are often complementary, and their combinations represent a powerful synergy. Finally, the lessons we learnt including the advantages and pitfalls of the described approaches are discussed.

Cardioprotective Effect of Novel Matrix Metalloproteinase Inhibitors.

BACKGROUND: We recently developed novel matrix metalloproteinase-2 (MMP-2) inhibitor small molecules for cardioprotection against ischemia/reperfusion injury and validated their efficacy in ischemia/reperfusion injury in cardiac myocytes. The aim of the present study was to test our lead compounds for cardioprotection in vivo in a rat model of acute myocardial infarction (AMI) in the presence or absence of hypercholesterolemia, one of the major comorbidities affecting cardioprotection. METHODS: Normocholesterolemic adult male Wistar rats were subjected to 30 min of coronary occlusion followed by 120 min of reperfusion to induce AMI. MMP inhibitors (MMPI)-1154 and -1260 at 0.3, 1, and 3 µmol/kg, MMPI-1248 at 1, 3, and 10 µmol/kg were administered at the 25th min of ischemia intravenously. In separate groups, hypercholesterolemia was induced by a 12-week diet (2% cholesterol, 0.25% cholic acid), then the rats were subjected to the same AMI protocol and single doses of the MMPIs that showed the most efficacy in normocholesterolemic animals were tested in the hypercholesterolemic animals. Infarct size/area at risk was assessed at the end of reperfusion in all groups by standard Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining, and myocardial microvascular obstruction (MVO) was determined by thioflavine-S staining. RESULTS: MMPI-1154 at 1 µmol/kg, MMPI-1260 at 3 µmol/kg and ischemic preconditioning (IPC) as the positive control reduced infarct size significantly; however, this effect was not seen in hypercholesterolemic animals. MVO in hypercholesterolemic animals decreased by IPC only. CONCLUSIONS: This is the first demonstration that MMPI-1154 and MMPI-1260 showed a dose-dependent infarct size reduction in an in vivo rat AMI model; however, single doses that showed the most efficacy in normocholesterolemic animals were abolished by hypercholesterolemia. The further development of these promising cardioprotective MMPIs should be continued with different dose ranges in the study of hypercholesterolemia and other comorbidities.

Design and Selection of Novel C1s Inhibitors by In Silico and In Vitro Approaches.

The complement system is associated with various diseases such as inflammation or auto-immune diseases. Complement-targeted drugs could provide novel therapeutic intervention against the above diseases. C1s, a serine protease, plays an important role in the CS and could be an attractive target since it blocks the system at an early stage of the complement cascade. Designing C1 inhibitors is particularly challenging since known inhibitors are restricted to a narrow bioactive chemical space in addition selectivity over other serine proteases is an important requirement. The typical architecture of a small molecule inhibitor of C1s contains an amidine (or guanidine) residue, however, the discovery of non-amidine inhibitors might have high value, particularly if novel chemotypes and/or compounds displaying improved selectivity are identified. We applied various virtual screening approaches to identify C1s focused libraries that lack the amidine/guanidine functionalities, then the in silico generated libraries were evaluated by in vitro biological assays. While 3D structure-based methods were not suitable for virtual screening of C1s inhibitors, and a 2D similarity search did not lead to novel chemotypes, pharmacophore model generation allowed us to identify two novel chemotypes with submicromolar activities. In three screening rounds we tested altogether 89 compounds and identified 20 hit compounds (<10 µM activities; overall hit rate: 22.5%). The highest activity determined was 12 nM (1,2,4-triazole), while for the newly identified chemotypes (1,3-benzoxazin-4-one and thieno[2,3-d][1,3]oxazin-4-one) it was 241 nM and 549 nM, respectively.

Inhibition of the LOX enzyme family members with old and new ligands. Selectivity analysis revisited.

Lysyl oxidase (LOX) enzymes as potential drug targets maintain constant attention in the therapy of fibrosis, cancer and metastasis. In order to measure the inhibitory activity of small molecules on the LOX enzyme family members a fluorometric activity screening method was developed. During assay validation, previously reported non-selective small inhibitor molecules (BAPN, MCP-1, thiram, disulfiram) were investigated on all of the major LOX enzymes. We confirmed that MCP-1, thiram, disulfiram are in fact pan-inhibitors, while BAPN inhibits only LOX-like enzymes (preferably LOX-like-protein-2, LOXL2) in contrast to the previous reports. We measured the LOX inhibitory profile of a small targeted library generated by 2D ligand-based chemoinformatics methods. Ten hits (10.4% hit rate) were identified, and the compounds showed distinct activity profiles. Potential inhibitors were also identified for LOX-like-protein-3 (LOXL3) and LOX-like-protein-4 (LOXL4), that are considered as emerging drug targets in the therapy of melanoma and gastric cancer.

Development of Matrix Metalloproteinase-2 Inhibitors for Cardioprotection.

The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 µM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

The Life of Pi Star: Exploring the Exciting and Forbidden Worlds of the Benzophenone Photophore.

The widespread applications of benzophenone (BP) photochemistry in biological chemistry, bioorganic chemistry, and material science have been prominent in both academic and industrial research. BP photophores have unique photochemical properties: upon n-π* excitation at 365 nm, a biradicaloid triplet state is formed reversibly, which can abstract a hydrogen atom from accessible C-H bonds; the radicals subsequently recombine, creating a stable covalent C-C bond. This light-directed covalent attachment process is exploited in many different ways: (i) binding/contact site mapping of ligand (or protein)-protein interactions; (ii) identification of molecular targets and interactome mapping; (iii) proteome profiling; (iv) bioconjugation and site-directed modification of biopolymers; (v) surface grafting and immobilization. BP photochemistry also has many practical advantages, including low reactivity toward water, stability in ambient light, and the convenient excitation at 365 nm. In addition, several BP-containing building blocks and reagents are commercially available. In this review, we explore the "forbidden" (transitions) and excitation-activated world of photoinduced covalent attachment of BP photophores by touring a colorful palette of recent examples. In this exploration, we will see the pros and cons of using BP photophores, and we hope that both novice and expert photolabelers will enjoy and be inspired by the breadth and depth of possibilities.

Combination of Pharmacophore Matching, 2D Similarity Search, and In Vitro Biological Assays in the Selection of Potential 5-HT6 Antagonists from Large Commercial Repositories.

Rapid in silico selection of target-focused libraries from commercial repositories is an attractive and cost-effective approach. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compound databases, but the generated library requires further focusing. We report here a combination of the 2D approach with pharmacophore matching which was used for selecting 5-HT6 antagonists. In the first screening round, 12 compounds showed >85% antagonist efficacy of the 91 screened. For the second-round (hit validation) screening phase, pharmacophore models were built, applied, and compared with the routine 2D similarity search. Three pharmacophore models were created based on the structure of the reference compounds and the first-round hit compounds. The pharmacophore search resulted in a high hit rate (40%) and led to novel chemotypes, while 2D similarity search had slightly better hit rate (51%), but lacking the novelty. To demonstrate the power of the virtual screening cascade, ligand efficiency indices were also calculated and their steady improvement was confirmed.

Combination of 2D/3D ligand-based similarity search in rapid virtual screening from multimillion compound repositories. Selection and biological evaluation of potential PDE4 and PDE5 inhibitors.

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost effective approach. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compound databases but the generated library requires further focusing by various 2D/3D chemoinformatics tools. We report here a combination of the 2D approach with a ligand-based 3D method (Screen3D) which applies flexible matching to align reference and target compounds in a dynamic manner and thus to assess their structural and conformational similarity. In the first case study we compared the 2D and 3D similarity scores on an existing dataset derived from the biological evaluation of a PDE5 focused library. Based on the obtained similarity metrices a fusion score was proposed. The fusion score was applied to refine the 2D similarity search in a second case study where we aimed at selecting and evaluating a PDE4B focused library. The application of this fused 2D/3D similarity measure led to an increase of the hit rate from 8.5% (1st round, 47% inhibition at 10 µM) to 28.5% (2nd round at 50% inhibition at 10 µM) and the best two hits had 53 nM inhibitory activities.

[Selection of a melanine concentrating hormone receptor-1 (MCHR1) antagonists' focused library and its biological screening with AequoScreen]. / Melanin koncentráló hormon receptor-1 (MCHR1) antagonista fókuszált könyvtár kiválasztása és in vitro biológiai szurése AequosCreen esszével.

Target focused libraries can be rapidly selected by 2D virtual screening methods from multimillion compounds' repositories if structures of active compounds are available. In the present study a multi-step virtual and in vitro screening cascade is reported to select Melanin Concentrating Hormone Receptor-1 (MCHR1) antagonists. The 2D similarity search combined with physicochemical parameter filtering is suitable for selecting candidates from multimillion compounds' repository. The seeds of the first round virtual screening were collected from the literature and commercial databases, while the seeds of the second round were the hits of the first round. In vitro screening underlined the efficiency of our approach, as in the second screening round the hit rate (8.6 %) significantly improved compared to the first round (1.9%), reaching the antagonist activity even below 10 nM.

A chemocentric approach to the identification of cancer targets.

A novel chemocentric approach to identifying cancer-relevant targets is introduced. Starting with a large chemical collection, the strategy uses the list of small molecule hits arising from a differential cytotoxicity screening on tumor HCT116 and normal MRC-5 cell lines to identify proteins associated with cancer emerging from a differential virtual target profiling of the most selective compounds detected in both cell lines. It is shown that this smart combination of differential in vitro and in silico screenings (DIVISS) is capable of detecting a list of proteins that are already well accepted cancer drug targets, while complementing it with additional proteins that, targeted selectively or in combination with others, could lead to synergistic benefits for cancer therapeutics. The complete list of 115 proteins identified as being hit uniquely by compounds showing selective antiproliferative effects for tumor cell lines is provided.

Combining 2D and 3D in silico methods for rapid selection of potential PDE5 inhibitors from multimillion compounds' repositories: biological evaluation.

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost-effective approach when starting new drug discovery projects. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compounds' databases. This in silico approach can be combined with physico-chemical parameter filtering based on the property space of the active compounds and 3D virtual screening if the structure of the target protein is available. A multi-step virtual screening procedure was developed and applied to select potential phosphodiesterase 5 (PDE5) inhibitors in real time. The combined 2D/3D in silico method resulted in the identification of 14 novel PDE5 inhibitors with <1 µMIC(50) values and the hit rate in the second in silico selection and in vitro screening round exceeded the 20%.

N-Acylhydrazones as inhibitors of PDE10A.

Cyclic nucleotide phosphodiesterases (PDEs) are represented by a large superfamily of enzymes. A series of hydrazone-based inhibitors was synthesized and shown to be novel, potent, and selective against PDE10A. Optimized compounds of this class were efficacious in animal models of schizophrenia and may be useful for the treatment of this disease.

Stepwise aromatic nucleophilic substitution in continuous flow. Synthesis of an unsymmetrically substituted 3,5-diamino-benzonitrile library.

Aromatic or heteroaromatic ring precursors with 2-3 identical functionalities are often used in sequential derivatization depending on the reactivity difference or the selective execution of the reaction such as nucleophilic aromatic substitution. Continuous flow chemistry offers an enhanced parameter space (pressure and temperature) with rapid parameter optimization that ensures selectivity in many cases. We developed a flow chemistry procedure to carry out a stepwise aromatic nucleophilic substitution of difluoro-benzenes having an activating group in meta position to the fluorines. The mono-aminated products were obtained in high yield and selectivity in an extremely short reaction time, while applying higher temperature, longer reaction zone (or time), and employing higher excess of another amine reactant, the subsequent introduction of the second amino group was also successfully achieved leading to an unsymmetrically substituted 3,5-diamino-benzonitrile library.

Matrix metalloproteinase inhibitors: a critical appraisal of design principles and proposed therapeutic utility.

Matrix metalloproteinases (MMPs) play an important role in tissue remodelling associated with various physiological and pathological processes, such as morphogenesis, angiogenesis, tissue repair, arthritis, chronic heart failure, chronic obstructive pulmonary disease, chronic inflammation and cancer metastasis. As a result, MMPs are considered to be viable drug targets in the therapy of these diseases. Despite the high therapeutic potential of MMP inhibitors (MMPIs), all clinical trials have failed to date, except for doxycycline for periodontal disease. This can be attributed to (i) poor selectivity of the MMPIs, (ii) poor target validation for the targeted therapy and (iii) poorly defined predictive preclinical animal models for safety and efficacy. Lessons from previous failures, such as recent discoveries of oxidative/nitrosative activation and phosphorylation of MMPs, as well as novel non-matrix related intra- and extracellular targets of MMP, give new hope for MMPI development for both chronic and acute diseases. In this article we critically review the major structural determinants of the selectivity and the milestones of past design efforts of MMPIs where 2-/3-dimensional structure-based methods were intensively applied. We also analyse the in vitro screening and preclinical/clinical pharmacology approaches, with particular emphasis on drawing conclusions on how to overcome efficacy and safety problems through better target validation and design of preclinical studies.