AutoPH4: An Automated Method for Generating Pharmacophore Models from Protein Binding Pockets.

Pharmacophore models are widely used in computational drug discovery (e.g., in the virtual screening of drug molecules) to capture essential information about interactions between ligands and a target protein. Generating pharmacophore models from protein structures is typically a manual process, but there has been growing interest in automated pharmacophore generation methods. Automation makes feasible the processing of large numbers of protein conformations, such as those generated by molecular dynamics (MD) simulations, and thus may help achieve the longstanding goal of incorporating protein flexibility into virtual screening workflows. Here, we present AutoPH4, a new automated method for generating pharmacophore models based on protein structures; we show that a virtual screening workflow incorporating AutoPH4 ranks compounds more accurately than any other pharmacophore-based virtual screening workflow for which results on a public benchmark have been reported. The strong performance of the virtual screening workflow indicates that the AutoPH4 component of the workflow generates high-quality pharmacophores, making AutoPH4 promising for use in future virtual screening workflows as well, such as ones that use conformations generated by MD simulations.

Discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as novel, highly potent and selective, orally bioavailable inhibitors of Tyrosine Threonine Kinase, TTK.

TTK/Mps1 is a key kinase controlling progression of cell division via participation in the mitotic spindle assembly checkpoint and is overexpressed in a number of human cancers. Herein we report the discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as a potent, novel class of TTK inhibitors. The series was identified by means of bioisosteric replacement of the related imidazopyrazine and imidazopyridazine scaffolds. Optimization led to the identification of compounds with excellent potency (Ki=0.8nM) and exceptional kinase selectivity. The SAR indicates a strong dependence of activity on the presence of the N-cyclopropyl-2-methylbenzamide moiety delineating the geometry for 1½ type kinase inhibitor. Molecular modeling indicates the extensive and optimal contacts, mediated through H-bonds and hydrophobic interactions, are responsible for the selectivity and potency of the inhibitors. The compounds demonstrate a strong anti-proliferative activity in a panel of human cancer cell lines (HCT116 GI50<15nM) and good rodent pharmacokinetics (oral %F 97%).

Design and optimization of (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones as potent PLK4 inhibitors with oral antitumor efficacy.

Previous efforts from our laboratory demonstrated that (E)-3-((3-(E)-vinylaryl)-1H-indazol-6-yl)methylene)-indolin-2-ones are potent PLK4 inhibitors with in vivo anticancer efficacy upon IP dosing. As part of a continued effort to develop selective and orally efficacious inhibitors, we examined variations on this theme wherein 'directly-linked' aromatics, pendant from the indazole core, replace the arylvinyl moiety. Herein, we describe the design and optimization of this series which was ultimately superseded by (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones. The latter compounds are potent and selective inhibitors of PLK4 with oral exposure in rodents and in vivo anticancer activity. Compound 13b, in particular, has a bioavailability of 22% and achieved a 96% tumor growth inhibition in an MDA-MB-468 xenograft study.

[Operation - rehabilitation - employment]. / Mutét - rehabilitáció - munkavállalás.

In this article we would like to bring the attention to the importance of early rehabilitation, coordinated operation of different subdivisions of rehabilitation and the expanding opportunities after medical care through following a 38- year-old female patient's case who was operated 6 years ago with anaplasticus oligodendroglioma. We find it important that the experts working on the field of health care, social services or labour should be aware of the possibilities of rehabilitation of their patients from the capability assessment, through its development until their placement in integrated labour market. It is important, that even during the medical care both the client and their relatives receive proper information.

Discovery of inhibitors of the mitotic kinase TTK based on N-(3-(3-sulfamoylphenyl)-1H-indazol-5-yl)-acetamides and carboxamides.

TTK kinase was identified by in-house siRNA screen and pursued as a tractable, novel target for cancer treatment. A screening campaign and systematic optimization, supported by computer modeling led to an indazole core with key sulfamoylphenyl and acetamido moieties at positions 3 and 5, respectively, establishing a novel chemical class culminating in identification of 72 (CFI-400936). This potent inhibitor of TTK (IC50=3.6nM) demonstrated good activity in cell based assay and selectivity against a panel of human kinases. A co-complex TTK X-ray crystal structure and results of a xenograft study with TTK inhibitors from this class are described.

Numerical errors in minimization based binding energy calculations.

This work examines the effect of small input perturbations on binding energies computed from differences between energy minimized structures, such as the Prime MM-GBSA and MOE MM-GB/VI methods. The applied perturbations include translations of the cognate ligand in the binding site by a maximum of 0.1 Å along each coordinate or the permutation of the order of atoms of the cognate ligand without any changes to the atom coordinates. These seemingly inconsequential input changes can lead to as much as 17 kcal/mol differences in the computed binding energy. The calculated binding energies cluster around discrete values, which correspond to specific ligand poses. It appears that the largest variations are observed for target-ligand systems in which there is a possibility for multiple poses with strong hydrogen bonds. The barriers between different poses can appear fractal-like, making it difficult to predict which solution will be produced from a given input. Including protein flexibility in MM-GBSA calculations further increases numerical instability, and the protein strain terms seem to be the major factor contributing to this sensitivity. In such calculations it appears unwise to extend the flexible region beyond 6 Å.

Numerical errors and chaotic behavior in docking simulations.

This work examines the sensitivity of docking programs to tiny changes in ligand input files. The results show that nearly identical ligand input structures can produce dramatically different top-scoring docked poses. Even changing the atom order in a ligand input file can produce significantly different poses and scores. In well-behaved cases the docking variations are small and follow a normal distribution around a central pose and score, but in many cases the variations are large and reflect wildly different top scores and binding modes. The docking variations are characterized by statistical methods, and the sensitivity of high-throughput and more precise docking methods are compared. The results demonstrate that part of docking variation is due to numerical sensitivity and potentially chaotic effects in current docking algorithms and not solely due to incomplete ligand conformation and pose searching. These results have major implications for the way docking is currently used for pose prediction, ranking, and virtual screening.

Automated generation of turn mimetics: proof of concept study for the MC4 receptor.

An algorithm has been devised for the automatic design of peptide turn mimetics, particularly applicable to peptide-activated GPCRs. The method is based on flexible alignments using a new design paradigm and scoring system that aims to reduce the molecular weight of the compound and preferentially lead to drug like molecules. The process can be applied either as a de novo design or a virtual screening tool. Its use has been demonstrated by the design of novel double digit nanomolar ligands for the melanocortin 4 receptor (MC4). The method is, in principle, applicable to any type of receptor, including orphan receptors.

Structural chemistry of the histone methyltransferases cofactor binding site.

Histone methyltransferases (HMTs) transfer a methyl group from the cofactor S-adenosyl methionine to lysine or arginine residues on histone tails, thereby regulating chromatin compaction, binding of effector proteins and gene transcription. HMTs constitute an emerging target class in diverse disease areas, and selective chemical probes are necessary for target validation. Potent and selective competitors of the substrate peptide have been reported, but the chemical tractability of the cofactor binding site is poorly understood. Here, a systematic analysis of this site across structures of 14 human HMTs or close homologues was conducted. The druggability, interaction hotspots, and diversity of the cofactor binding pocket were dissected. This analysis strongly suggests that this site is chemically tractable. General principles underlying tight binding and specific guidelines to achieve selective inhibition are presented.

Reducing docking score variations arising from input differences.

The variability of docking results as a function of variations in ligand input conformations was studied for the GOLD, Glide, FlexX, and Surflex programs. It is concluded that there are two major effects leading to such variability: the adequacy of conformational search during docking and random "chaotic" effects arising from sensitivity to small input perturbations. It is shown that although the former is generally the stronger effect, the latter is also highly significant for almost all docking engines. The strong target-to-target variation of the magnitude of these effects is emphasized. The performance of different packages is compared using these measures. Guidelines are provided for different programs to reduce variability and improve reproducibility, which involve using a small number of input conformations as starting points for docking, followed by the selection of the top scoring docked pose from the results as the best docked solution.

Fragment Hits: What do They Look Like and How do They Bind?

A "fragment hit", a molecule of low molecular weight that has been validated to bind to a target protein, can be an effective chemical starting point for a drug discovery project. Our ability to find and progress fragment hits could potentially be improved by enhancing our understanding of their binding properties, which to date has largely been based on tacit knowledge and reports from individual projects. In the work reported here, we systematically analyzed the molecular and binding properties of fragment hits using 489 published protein-fragment complexes. We identified a number of notable features that these hits tend to have in common, including preferences in buried surface area upon binding, hydrogen bonding and other directional interactions with the protein targets, structural topology, functional-group occurrence, and degree of carbon saturation. In the future, taking account of these preferences in designing and selecting fragments to screen against protein targets may increase the chances of success in fragment screening campaigns.

The use of ligand-based de novo design for scaffold hopping and sidechain optimization: two case studies.

This paper describes the application of de novo design utilizing exclusively ligand information. In the current approach, ligand design criteria, including pharmacophores, similarity and desired properties are applied as part of a fitness function driving the design process, instead of using them as filters after the process. This allows relevant parts of chemical space to be explored more efficiently. Two case studies of successful ligand design are also presented, one aimed at scaffold hopping, the other for exploring substitution patterns around a novel scaffold.

[Treatment of spastic upper limb with botulinum toxin]. / A felso végtagi spasticitas kezelése botulinustoxinnal.

OBJECTIVE: Examination of the effect of local botulinum toxin treatment on spastic upper limb, in patients with various brain injuries. PATIENTS AND METHOD: Prospective study at the Traumatic Brain Injury Rehabilitation Unit of the National Institute for Medical Rehabilitation in the year 2003 and 2004. Thirteen patients (eight with stroke and five with traumatic brain injury) were treated locally on the spastic upper limb with 100 units botulinum A toxin. RESULTS: Spasticity decreased one or two level on Modified Ashworth Scale, and in nine cases the good result were observed still at the end of 3rd month. No local or other complication was detected. CONCLUSIONS: Local treatment with botulinum toxin is an effective and safe method to decrease spasticity on upper limb in patients with various brain injuries.

Consensus scoring for protein-ligand interactions.

This article reviews the application of consensus scoring for cases when the target 3D structure is known. Comparing the performance of different methods is not a trivial task, and it appears that consensus scoring usually substantially improves virtual screening performance, contributing to better enrichments. It also seems to improve--albeit less dramatically--the prediction of bound conformations and poses. The prediction of binding energies is still rather inaccurate and although consensus scoring generally improves these predictions, more development is required before it can be used for this purpose in routine lead optimization.

Discovery of Pyrazolo[1,5-a]pyrimidine TTK Inhibitors: CFI-402257 is a Potent, Selective, Bioavailable Anticancer Agent.

This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1(1)/2 type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.

Administration of estramustine in response to changes in the prostate-specific antigen and Karnofsky index in the treatment of prostate cancer.

Androgen ablation is palliative and does not cure advanced prostate cancer. The hormone-sensitive cells die and the hormone-resistant cells overgrow, resulting in disease progression. The drug of choice for secondary treatment is estramustine (Estracyt). The success of the therapy is followed by changes of the prostate-specific antigen level and Karnofsky scale. In the present study, the results of estramustine treatment of 79 patients with advanced prostate cancer in 12 hospitals were evaluated. The mean prostate-specific antigen level improved for 6 months, but rose from the ninth month on. The improvement in the subjective condition of the patients paralleled the change in the prostate-specific antigen level. The short time of improvement was a consequence of the very high prostate-specific antigen level and the poor general condition. Estramustine administration is recommended when the prostate-specific antigen level becomes more than doubled following primary treatment. At a starting prostate-specific antigen level of > 100 ng/ml, the treatment leads to total androgen blockade. If the prostate-specific antigen level has not decreased after treatment for 3 months, the secondary strategy is to apply chemotherapy.

[The place of estramustine in the treatment of prostate cancer]. / Az estramustin helye a prostatarák kezelésében.

INTRODUCTION/AIMS: Prostate cancer is a dynamic disease. Androgen ablation is palliative, and does not cure advanced prostate cancer. The hormone-sensitive cells die, and the hormone-resistant cells come into excess; the disease then progresses, which results in a deterioration of the condition of the patient. The theoretical basis of the curing strategy is the fact that the prostate tumour itself changes during the progression; the molecular determinants of the resistance are present in the varying stages of the disease. The treatment of advanced prostate cancer remains unsolved; it is a well-known fact that a hormone-resistant state develops after the primary treatment forms (androgen withdrawal). The drug of choice for the secondary treatment is estramustine. This can be utilized as monotherapy or in combination. METHODS: In the present study, the results of estramustine treatment of 79 patients with advanced prostate cancer were evaluated. The preparation, known and clinically applied for more than 20 years, was studied in 12 centres. RESULTS: The mean prostate-specific antigen level improved for 6 months, but rose from the 9th month on. The improvement in the subjective condition of the patients paralleled the change in the prostate-specific antigen level. The shortness of the improvement was a consequence of the very high prostate-specific antigen level and the poor general condition. CONCLUSIONS: Estramustine administration is recommended when the prostate-specific antigen level becomes more than doubled following the primary treatment. At a starting prostate-specific antigen level of >100 ng/ml, the treatment leads to total androgen blockade. If the prostate-specific antigen level has not decreased after treatment for 3 months, the secondary strategy is to apply chemotherapy.

The discovery of Polo-like kinase 4 inhibitors: design and optimization of spiro[cyclopropane-1,3'[3H]indol]-2'(1'H).ones as orally bioavailable antitumor agents.

Polo-like kinase 4 (PLK4), a unique member of the polo-like kinase family of serine-threonine kinases, is a master regulator of centriole duplication that is important for maintaining genome integrity. Overexpression of PLK4 is found in several human cancers and is linked with a predisposition to tumorigenesis. Previous efforts to identify potent and efficacious PLK4 inhibitors resulted in the discovery of (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones, which are superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones reported herein. Optimization of this new cyclopropane-linked series was based on a computational model of a PLK4 X-ray structure and SAR attained from the analogous alkenelinked series. The racemic cyclopropane-linked compounds showed PLK4 affinity and antiproliferative activity comparable to their alkene-linked congeners with improved hysicochemical, ADME, and pharmacokinetic properties. Positive xenograft results from the MDA-MB-468 human breast cancer xenograft model for compound 18 support the investigation of PLK4 inhibitors as anticancer therapeutics. A PLK4 X-ray co-structure with racemate 18 revealed preferential binding of the 1R,2S enantiomer to the PLK4 kinase domain.

The discovery of Polo-like kinase 4 inhibitors: identification of (1R,2S).2-(3-((E).4-(((cis).2,6-dimethylmorpholino)methyl)styryl). 1H.indazol-6-yl)-5'-methoxyspiro[cyclopropane-1,3'-indolin]-2'-one (CFI-400945) as a potent, orally active antitumor agent.

Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1Hindazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This work reports the discovery of a novel one-pot double SN2 displacement reaction for the stereoselective installation of the desired asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth. We conclude with the identification of compound 48 (designated CFI-400945) as a novel clinical candidate for cancer therapy.

The Discovery of Orally Bioavailable Tyrosine Threonine Kinase (TTK) Inhibitors: 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides as Anticancer Agents.

The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, are not drug-like. By eliminating the polar 3-sulfonamide group and grafting a heterocycle at the 4 position of the phenyl ring, potent inhibitors with oral exposure were obtained. An X-ray cocrystal structure and a refined binding model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors, namely 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides. Compounds incorporating the 3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl bicyclic system were potent (TTK IC50 < 10 nM, HCT116 GI50 < 0.1 µM), displayed low off-target activity (>500×), and microsomal stability (T(1/2) > 30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer. Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon oral dosing, and was selected for preclinical evaluation.