Tetrahydroprotoberberine alkaloids with dopamine and σ receptor affinity.

Two series of analogues of the tetrahydroprotoberberine (THPB) alkaloid (±)-stepholidine that (a) contain various alkoxy substituents at the C10 position and, (b) were de-rigidified with respect to (±)-stepholidine, were synthesized and evaluated for affinity at dopamine and σ receptors in order to evaluate effects on D3 and σ2 receptor affinity and selectivity. Small n-alkoxy groups are best tolerated by D3 and σ2 receptors. Among all compounds tested, C10 methoxy and ethoxy analogues (10 and 11 respectively) displayed the highest affinity for σ2 receptors as well as σ2 versus σ1 selectivity and also showed the highest D3 receptor affinity. De-rigidification of stepholidine resulted in decreased affinity at all receptors evaluated; thus the tetracyclic THPB framework is advantageous for affinity at dopamine and σ receptors. Docking of the C10 analogues at the D3 receptor, suggest that an ionic interaction between the protonated nitrogen atom and Asp110, a H-bond interaction between the C2 phenol and Ser192, a H-bond interaction between the C10 phenol and Cys181 as well as hydrophobic interactions of the aryl rings to Phe106 and Phe345, are critical for high affinity of the compounds.

Synthesis and evaluation of aporphine analogs containing C1 allyl isosteres at the h5-HT(2A) receptor.

A series of C1 aporphine analogs related to compound 5 and that contain substituted allylic, alkynyl, nitrile, ester and benzyl groups was synthesized and evaluated for affinity at h5HT2A and α1A receptors in functional activity assays that measure calcium release. The presence of branched allylic substituent groups diminished affinity for the h5HT2A receptor. Likewise, the alkynyl, nitrile and ester derivatives evaluated displayed lower 5-HT2A receptor affinity as compared to 5. Hydrophobic, steric and electronic effects impact the affinity of p-substituted benzyl derivatives 8i-8k but in different ways. High hydrophobicity and size favor 5-HT2A affinity whereas, high electronegativity disfavors 5-HT2A affinity. p-Bromobenzyl analog 8k was identified as a 5-HT2A receptor selective ligand, with the highest 5-HT2A receptor affinity of any aporphine known to date. Most of the other analogs were selective for the 5-HT2A versus the α1A receptor. ChemScore binding energies from docking studies correlated qualitatively with the observed trends in affinity for 8i-8k, although the binding energies were not well differentiated quantitatively.

Successful treatment of immune checkpoint inhibitor-related periaortitis.

We report a 64-year-old patient with melanoma receiving ipilimumab and nivolumab therapy who presented with a periaortic soft tissue mass around the abdominal aorta on restaging fluorodeoxyglucose positron emission tomography/computed tomography imaging. Clinical, laboratory, and radiologic findings resulted in a diagnosis of immune checkpoint inhibitor-related periaortitis. Periaortitis is a rare disease presenting with fibro-inflammatory tissue around the aorta and may lead to serious complications. Immune checkpoint inhibitors were discontinued, and the patient was treated with glucocorticoids, leading to a complete resolution of the periaortitis. To our knowledge, this is only the third reported case of immune checkpoint inhibitor-related periaortitis.

Prostate-specific Membrane Antigen: Diagnostics.

Since its clinical introduction in May 2011, prostate-specific membrane antigen (PSMA)-PET/computed tomography has quickly gained worldwide recognition as a significant breakthrough in prostate cancer diagnostics. In the meantime, several new PSMA radioligands for PET imaging have been introduced into routine clinical practice. This article aims to introduce the most commonly used tracers and their key areas of application.

Tumor microenvironment mechanisms and bone metastatic disease progression of prostate cancer.

During disease progression from primary towards metastatic prostate cancer (PCa), and in particular bone metastases, the tumor microenvironment (TME) evolves in parallel with the cancer clones, altering extracellular matrix composition (ECM), vasculature architecture, and recruiting specialized tumor-supporting cells that favor tumor spread and colonization at distant sites. We introduce the clinical profile of advanced metastatic PCa in terms of common genetic alterations. Findings from recently developed models of PCa metastatic spread are discussed, focusing mainly on the role of the TME (mainly matrix and fibroblast cell types), at distinct stages: premetastatic niche orchestrated by the primary tumor towards the metastatic site and bone metastasis. We report evidence of premetastatic niche formation, such as the mechanisms of distant site conditioning by extracellular vesicles, chemokines and other tumor-derived mechanisms, including altered cancer cell-ECM interactions. Furthermore, evidence supporting the similarities of stroma alterations among the primary PCa and bone metastasis, and contribution of TME to androgen deprivation therapy resistance are also discussed. We summarize the available bone metastasis transgenic mouse models of PCa from a perspective of pro-metastatic TME alterations during disease progression and give an update on the current diagnostic and therapeutic radiological strategies for bone metastasis clinical management.

Prostate Cancer Theranostics: From Target Description to Imaging.

Prostate-specific membrane antigen-PET/computed tomography (PSMA-PET/CT) is the investigation of choice for imaging prostate cancer. Demonstrating high diagnostic accuracy, PSMA-PET/CT detects disease at very early stages of recurrence, where the chances of a definitive cure may be at their greatest. A number of PSMA-radioligands are in established clinical routine, and there are currently only limited data and no single tracer can clearly be advocated over the others at present. Further clinical trial data, comparing and contrasting radiotracers and reporting outcome-based data are necessary to further increase the implementation of this very promising imaging modality.

Prostate Cancer Theranostics: PSMA Targeted Therapy.

Prostate-specific membrane antigen (PSMA) has been the subject of numerous studies within the last 3 decades. PSMA-targeted imaging and therapy have significantly changed the management of patients with prostate cancer in various disease stages, especially in advanced metastasized castration-resistant prostate cancer. Lutetium-177-conjugated PSMA-617 or PSMA-I&T (Lu-PSMA) has shown promising results in multicenter retrospective and monocenter prospective trials. The aim of this review is to provide an overview of the history and current and future developments of PSMA-targeted therapy. A special focus of this review is on PSMA PET-guided management of patients receiving PSMA-targeted radioligand therapy.

Synthesis and evaluation of C9 alkoxy analogues of (-)-stepholidine as dopamine receptor ligands.

Tetrahydroprotoberberine alkaloids have shown interesting polypharmacological actions at dopamine receptors and are a unique template from which to mine novel molecules with dual selective actions at D1 and D3 receptors. Such compounds will be valuable to evaluate as anti-cocaine therapeutics. Towards that eventual goal, we engaged an SAR study in which a series of C9 alkoxy analogues of the D1/D2/D3 ligand (-)-stepholidine that possessed or lacked a C12 bromo functionality, were synthesized and evaluated for affinity at dopamine D1, D2 and D3 receptors. We found that the analogues are generally selective for the D1 receptor. Small n-alkoxy substituents (up to 4 carbons in length) were generally well tolerated for high D1 affinity but such groups reduced D3 affinity. In the case of C12 brominated analogues, C9 alkoxylation also had little effect on D1 affinity for the smaller alkoxy groups, but reduced D2 and D3 affinities significantly. C12 bromination tends to increase D1 receptor selectivity. A number of compounds were identified that retain affinity for D1 and D3 receptors but lack D2 receptor affinity. Among them, compound 22a was found to be a selective D1/D3 dual antagonist (Ki = 5.3 and 106 nM at D1 and D3 receptors). Docking studies performed on the analogues at the D3 receptor revealed a number of interactions that are important for affinity including a critical N - Asp110 salt bridge motif, H-bonds to Ser192 and Cys181 and hydrophobic interactions between the aryl rings and Phe106 and Phe345. The analogues adopt an orientation in which ring A is located in the orthosteric binding site while the C9 alkoxy substituents attached to ring D project into the secondary binding pocket of the D3 receptor.

Receptor flexibility in de novo ligand design and docking.

One of the major problems in computational drug design is incorporation of the intrinsic flexibility of protein binding sites. This is particularly crucial in ligand binding events, when induced fit can lead to protein structure rearrangements. As a consequence of the huge conformational space available to protein structures, receptor flexibility is rarely considered in ligand design procedures. In this work, we present an algorithm for integrating protein binding-site flexibility into de novo ligand design and docking processes. The approach allows dynamic rearrangement of amino acid side chains during the docking and design simulations. The impact of protein conformational flexibility is investigated in the docking of highly active inhibitors in the binding sites of acetylcholinesterase and human collagenase (matrix metalloproteinase-1) and in the design of ligands in the S1' pocket of MMP-1. The results of corresponding simulations for both rigid and flexible binding sites are compared in order to gauge the influence of receptor flexibility in drug discovery protocols.

Receptor flexibility in the in silico screening of reagents in the S1' pocket of human collagenase.

A major difficulty in structure-based molecular design is the prediction of the structure of the protein-ligand complex because of the enormous number of degrees of freedom. Commonly, the target protein is kept rigid in a single low-energy conformation. However, this does not reflect the dynamic nature of protein structures. In this work, we investigate the influence of receptor flexibility in virtual screening of reagents on a common scaffold in the S1' pocket of human collagenase (matrix metalloproteinase-1). We compare screening using a single-crystal structure and multiple NMR structures, both apo and holo forms. We also investigate two computational methods of addressing receptor flexibility that can be used when NMR data are not available. The results from virtual screening using the experimental structures are compared to those obtained using the two computational methods. From the results, we draw conclusions about the impact of target flexibility on the identification of active and diverse reagents in a virtual screening protocol.

Molecular Structures of Methyldifluoroarsine, CH(3)AsF(2), and Dimethylfluoroarsine, (CH(3))(2)AsF, in the Gas Phase As Determined by Electron Diffraction and ab InitioCalculations.

The structures of gaseous CH(3)AsF(2) and (CH(3))(2)AsF have been determined by electron diffraction incorporating vibrational amplitudes derived from ab initio force fields scaled by experimental frequencies and, for the difluoride, restrained by microwave constants. The following parameters (r(alpha) degrees structure, distances in pm, angles in degrees) have been determined for CH(3)AsF(2): r(As-C) = 194.6(4), r(As-F) = 173.1(1), angleCAsF = 95.2(1), angleFAsF = 97.0(1). For (CH(3))(2)AsF structural refinement gives r(As-C) = 195.1(1), r(As-F) = 175.4(1), angleCAsF = 95.3(5), and angleCAsC = 96.9(8). For the series (CH(3))(3)As, (CH(3))(2)AsF, CH(3)AsF(2), and AsF(3), both As-C and As-F bond lengths are shortened with increasing numbers of F atoms, but the angles CAsF and FAsF are almost invariant.

DNA polymerase beta catalysis: are different mechanisms possible?

DNA polymerases are crucial constituents of the complex cellular machinery for replicating and repairing DNA. Discerning mechanistic pathways of DNA polymerase on the atomic level is important for revealing the origin of fidelity discrimination. Mammalian DNA polymerase beta (pol beta), a small (39 kDa) member of the X-family, represents an excellent model system to investigate polymerase mechanisms. Here, we explore several feasible low-energy pathways of the nucleotide transfer reaction of pol beta for correct (according to Watson-Crick hydrogen bonding) G:C basepairing versus the incorrect G:G case within a consistent theoretical framework. We use mixed quantum mechanics/molecular mechanics (QM/MM) techniques in a constrained energy minimization protocol to effectively model not only the reactive core but also the influence of the rest of the enzymatic environment and explicit solvent on the reaction. The postulated pathways involve initial proton abstraction from the terminal DNA primer O3'H group, nucleophilic attack that extends the DNA primer chain, and elimination of pyrophosphate. In particular, we analyze several possible routes for the initial deprotonation step: (i) direct transfer to a phosphate oxygen O(Palpha) of the incoming nucleotide, (ii) direct transfer to an active site Asp group, and (iii) transfer to explicit water molecules. We find that the most probable initial step corresponds to step (iii), involving initial deprotonation to water, which is followed by proton migration to active site Asp residues, and finally to the leaving pyrophosphate group, with an activation energy of about 15 kcal/mol. We argue that initial deprotonation steps (i) and (ii) are less likely as they are at least 7 and 11 kcal/mol, respectively, higher in energy. Overall, the rate-determining step for both the correct and the incorrect nucleotide cases is the initial deprotonation in concert with nucleophilic attack at the phosphate center; however, the activation energy we obtain for the mismatched G:G case is 5 kcal/mol higher than that of the matched G:C complex, due to active site structural distortions. Taken together, our results support other reported mechanisms and help define a framework for interpreting nucleotide specificity differences across polymerase families, in terms of the concept of active site preorganization or the so-called "pre-chemistry avenue".

QUASI: a novel method for simultaneous superposition of multiple flexible ligands and virtual screening using partial similarity.

The structure of many receptors is unknown, and only information about diverse ligands binding to them is available. A new method is presented for the superposition of such ligands, derivation of putative receptor site models and utilization of the models for screening of compound databases. In order to generate a receptor model, the similarity of all ligands is optimized simultaneously taking into account conformational flexibility and also the possibility that the ligands can bind to different regions of the site and only partially overlap. Ligand similarity is defined with respect to a receptor site model serving as a common reference frame. The receptor model is dynamic and coevolves with the ligand alignment until an optimal self-consistent superposition is achieved. When ligand conformational flexibility is permitted, different superposition models are possible and consistent with the data. Clustering of the superposition solutions is used to obtain diverse models. When the models are used to screen a database of compounds, high enrichments are obtained, comparable to those obtained in docking studies.

The influence of variations of ligand protonation and tautomerism on protein-ligand recognition and binding energy landscape.

We investigate the influence of variations of ligand protonation and tautomeric states on the protein-ligand binding energy landscape by applying the concept of structural consensus. In docking simulations, allowing full flexibility of the ligand, we explore whether the native binding mode could be successfully recovered using a non-native ligand protonation state. Here, we consider three proteins, dihydrofolate reductase, transketolase, and alpha-trichosanthin, complexed with ligands having multiple tautomeric forms. We find that for the majority of protonation and tautomeric states the native binding mode can be recovered without a great loss of accuracy.

Exhaustive de novo design of low-molecular-weight fragments against the ATP-binding site of DNA-gyrase.

We present a de novo design approach to generating small fragments in the DNA-gyrase ATP-binding site using the computational drug design platform SkelGen. We have generated an exhaustive number of structural possibilities, which were subsequently filtered for site complementarity and synthetic tractability. A number of known active fragments are found, but most of the species created are potentially novel and could be valuable for further elaboration and development into lead-like structures.

Combinatorial ligand design targeted at protein families.

We describe a method to create ligands specific for a given protein family. The method is applied to generate ligand candidates for the cyclin-dependent kinase (CDK) family. The CDK family of proteins is involved in regulating the cell cycle by alternately activating and deactivating the cell's progression through the cycle. CDKs are activated by association with cyclin and are inhibited by complexation with small molecules. X-ray crystal structures are available for three of the thirteen known CDK family members: CDK2, CDK5 and CDK 6. In this work, we use novel computational approaches to design ligand candidates that are potentially inhibitory across the three CDK family members as well as more specific molecules which can potentially inhibit one or any combination of two of the three CDK family members. We define a new scoring term, SpecScore, to quantify the potential inhibitory power of the generated structures. According to a search of the World Drug Alerts, the highest scoring SpecScore molecule that is specific for the three CDK family members shows very similar chemical characteristics and functional groups to numerous molecules known to deactivate several members of the CDK family.

The effect of tightly bound water molecules on the structural interpretation of ligand-derived pharmacophore models.

The importance of the consideration of water molecules in the structural interpretation of ligand-derived pharmacophore models is explored. We compare and combine results from recently introduced methods for bound-water molecule identification in protein binding sites and ligand-superposition-based pharmacophore derivation, for the interpretation of ligand-derived pharmacophore models. In the analysis of thymidine kinase (HSV-1) and poly (ADP-ribose) polymerase (PARP), the concurrent application of both methods leads to an agreement in the prediction of tightly bound water molecules as key pharmacophoric points in the binding site of these proteins. This agreement has implications for approaching binding site analysis and consensus drug design, as it highlights how pharmacophore-based models of binding sites can include interaction features not only with protein groups but also with bound water molecules.

On the molecular pathology of neurodegeneration in IMPDH1-based retinitis pigmentosa.

Retinitis pigmentosa (RP), the hereditary degenerative disease of the photoreceptor neurons of the retina, probably represents the most prevalent cause of registered blindness amongst those of working age in developed countries. Mutations within the gene encoding inosine monophosphate dehydrogenase 1 (IMPDH1), the widely expressed rate-limiting enzyme of the de novo pathway of guanine nucleotide biosynthesis, have recently been shown to cause the RP10 form of autosomal dominant RP. We examined the expression of IMPDH1, IMPDH2 and HPRT transcripts, encoding enzymes of the de novo and salvage pathways of guanine nucleotide biosynthesis, respectively, in retinal sections of mice, the data indicating that the bulk of GTP within photoreceptors is generated by IMPDH1. Impdh1(-/-) null mice are shown here to display a slowly progressive form of retinal degeneration in which visual transduction, analysed by electroretinographic wave functions, becomes gradually compromised, although at 12 months of age most photoreceptors remain structurally intact. In contrast, the human form of RP caused by mutations within the IMPDH1 gene is a severe autosomal dominant degenerative retinopathy in those families that have been examined to date. Expression of mutant IMPDH1 proteins in bacterial and mammalian cells, together with computational simulations, indicate that protein misfolding and aggregation, rather than reduced IMPDH1 enzyme activity, is the likely cause of the severe phenotype experienced by human subjects. Taken together, these findings suggest that RP10 may represent an attractive target for therapeutic intervention, based upon a strategy combining simultaneous suppression of transcripts from normal and mutant IMPDH1 alleles with supplementation of GTP within retinal tissues.