Nonconscious information can be identified as task-relevant but not prioritized in working memory.

Two critical features of working memory are the identification and appropriate use of task-relevant information while avoiding distraction. Here, in 3 experiments, we explored if these features can be achieved also for nonconscious stimuli. Participants performed a delayed match-to-sample task in which task relevance of 2 competing stimuli was indicated by a cue, and continuous flash suppression was used to manipulate the conscious/nonconscious visual experience. Experiment 1 revealed better-than-chance performance with nonconscious stimuli, demonstrating goal-directed use of nonconscious task-relevant information. Experiment 2 demonstrated that the cue that defined task relevance must be conscious to allow such goal-directed use. In Experiment 3, multi-voxel pattern analyses of brain activity revealed that only the target was prioritized and maintained during conscious trials. Conversely, during nonconscious trials, both target and distractor were maintained. However, decoding of task relevance during the probe/test phase demonstrated identification of both target and distractor information. These results show that identification of task-relevant information can operate also on nonconscious material. However, they do not support the prioritization of nonconscious task-relevant information, thus suggesting a mismatch in the attentional mechanisms involved during conscious and nonconscious working memory.

Neural Evidence for Non-conscious Working Memory.

Recent studies have found that non-consciously perceived information can be retained for several seconds, a feat that has been attributed to non-conscious working memory processes. However, these studies have mainly relied on subjective measures of visual experience, and the neural processes responsible for non-conscious short-term retention remains unclear. Here we used continuous flash suppression to render stimuli non-conscious in a delayed match-to-sample task together with fMRI to investigate the neural correlates of non-conscious short-term (5-15 s) retention. The participants' behavioral performance was at chance level when they reported no visual experience of the sample stimulus. Critically, multivariate pattern analyses of BOLD signal during the delay phase could classify presence versus absence of sample stimuli based on signal patterns in frontal cortex, and its spatial position based on signal patterns in occipital cortex. In addition, univariate analyses revealed increased BOLD signal change in prefrontal regions during memory recognition. Thus, our findings demonstrate short-term maintenance of information presented non-consciously, defined by chance performance behaviorally. This non-consciously retained information seems to rely on persistent neural activity in frontal and occipital cortex, and may engage further cognitive control processes during memory recognition.

Intrinsic Clearance Assay Incubational Binding: A Method Comparison.

The fraction of unbound drug (fuinc) in in vitro intrinsic clearance (CLint) incubation is an important parameter in the pursuit of accurate clearance predictions and is often predicted using algorithms based on drug lipophilicity measures. However, analysis of an AstraZeneca database suggests that simple lipophilicity alone is a relatively poor predictor of fuinc measured using equilibrium dialysis. He fuinc value can also be measured directly in CLint assays using multiple concentrations of hepatocytes or microsomal protein. Since this approach informs of the unbound drug concentration in the assay used to predict in vivo clearance, it should be considered the gold standard method. As a starting point for building better predictive algorithms we aimed to determine if equilibrium dialysis really is an appropriate assay for assessing fuinc Employing a large number of compounds with a wide range of lipophilicities, experiments were performed to measure fuinc using rat hepatocytes (RH) and human liver microsomes (HLM) in both assay formats. A high percentage (94% and 93% for HLM and RH, respectively) of the fuinc values were within 2-fold when the compound distribution coefficient describing the ratio of compound concentration in octanol and pH 7.4 buffer when the test system is at equilibrium (lipophilicity measure) (logD7.4) values were less than 3.5. However, with logD7.4 values greater than these, the agreement was considerably worse. Additional experimental data generated indicated that this discrepancy was likely due to failings in the direct method when drug binding is high. Thus, we conclude that unbound CLint can be indeed calculated indirectly by incorporating equilibrium dialysis data with measured CLint but that simple lipophilicity descriptors alone may be inadequate for predicting fuinc.

Identification of Novel Series of Potent and Selective Relaxin Family Peptide Receptor 1 (RXFP1) Agonists.

Relaxin H2 is a clinically relevant peptide agonist for relaxin family peptide receptor 1 (RXFP1), but a combination of this hormone's short plasma half-life and the need for injectable delivery limits its therapeutic potential. We sought to overcome these limitations through the development of a potent small molecule (SM) RXFP1 agonist. Although two large SM HTS campaigns failed in identifying suitable hit series, we uncovered novel chemical space starting from the only known SM RXFP1 agonist series, represented by ML290. Following a design-make-test-analyze strategy based on improving early dose to man ranking, we discovered compound 42 (AZ7976), a highly selective RXFP1 agonist with sub-nanomolar potency. We used AZ7976, its 10 000-fold less potent enantiomer 43 and recombinant relaxin H2 to evaluate in vivo pharmacology and demonstrate that AZ7976-mediated heart rate increase in rats was a result of RXFP1 agonism. As a result, AZ7976 was selected as lead for continued optimization.

Discovery of Clinical Candidate AZD5462, a Selective Oral Allosteric RXFP1 Agonist for Treatment of Heart Failure.

Optimization of the highly potent and selective, yet metabolically unstable and poorly soluble hRXFP1 agonist AZ7976 led to the identification of the clinical candidate, AZD5462. Assessment of RXFP1-dependent cell signaling demonstrated that AZD5462 activates a highly similar panel of downstream pathways as relaxin H2 but does not modulate relaxin H2-mediated cAMP second messenger responsiveness. The therapeutic potential of AZD5462 was assessed in a translatable cynomolgus monkey heart failure model. Following 8 weeks of treatment with AZD5462, robust improvements in functional cardiac parameters including LVEF were observed at weeks 9, 13, and 17 without changes in heart rate or mean arterial blood pressure. AZD5462 was well tolerated in both rat and cynomolgus monkey and has successfully completed phase I studies in healthy volunteers. In summary, AZD5462 is a small molecule pharmacological mimetic of relaxin H2 signaling at RXFP1 and holds promise as a potential therapeutic approach to treat heart failure patients.

Less cortical complexity in ventromedial prefrontal cortex is associated with a greater preference for risky and immediate rewards.

In our everyday lives, we are often faced with situations in which we have to make choices that involve risky or delayed rewards. However, the extent to which we are willing to accept larger risky (over smaller certain) or larger delayed (over smaller immediate) rewards vary across individuals. Here we investigated the relationship between cortical surface complexity in medial prefrontal cortex and individual differences in risky and intertemporal preferences. We found that lower cortical complexity in ventromedial prefrontal cortex (vmPFC) was associated with a greater preference for risky and immediate rewards. In addition to these common structural associations in mPFC, we also found associations between lower cortical complexity and a greater preference for immediate rewards that extended into left dorsomedial prefrontal cortex and right vmPFC. Taken together, the shared association suggests that lower cortical complexity in vmPFC may be a structural marker for individual differences in impulsive behavior.

Neural and behavioral signatures of the multidimensionality of manipulable object processing.

Understanding how we recognize objects requires unravelling the variables that govern the way we think about objects and the neural organization of object representations. A tenable hypothesis is that the organization of object knowledge follows key object-related dimensions. Here, we explored, behaviorally and neurally, the multidimensionality of object processing. We focused on within-domain object information as a proxy for the decisions we typically engage in our daily lives - e.g., identifying a hammer in the context of other tools. We extracted object-related dimensions from subjective human judgments on a set of manipulable objects. We show that the extracted dimensions are cognitively interpretable and relevant - i.e., participants are able to consistently label them, and these dimensions can guide object categorization; and are important for the neural organization of knowledge - i.e., they predict neural signals elicited by manipulable objects. This shows that multidimensionality is a hallmark of the organization of manipulable object knowledge.

Disentangling hand and tool processing: Distal effects of neuromodulation.

Neural processing within a local brain region that responds to more than one object category (e.g., hands and tools) nonetheless have different functional connectivity patterns with other distal brain areas, which suggests that local processing can affect and/or be affected by processing in distal areas, in a category-specific way. Here we wanted to test whether administering either a hand- or tool-related training task in tandem with transcranial direct current stimulation (tDCS) to a region that responds both to hands and tools (posterior middle temporal gyrus; pMTG), modulated local and distal neural processing more for the trained than the untrained category in a subsequent fMRI task. After each combined tDCS/training session, participants viewed images of tools, hands, and animals, in an fMRI scanner. Using multivoxel pattern analysis, we found that tDCS stimulation to pMTG indeed improved the classification accuracy between tools vs. animals, but only when combined with a tool and not a hand training task. Surprisingly, tDCS stimulation to pMTG also improved classification accuracy between hands vs. animals when combined with a tool but not a hand training task. Our findings suggest that overlapping but functionally-specific networks may be engaged separately by using a category-specific training task together with tDCS - a strategy that can be applied more broadly to other cognitive domains using tDCS. By hypothesis, these effects on local processing are a direct result of within-domain connectivity constraints from domain-specific networks that are at play in the processing and organization of object representations.

Discovery by Virtual Screening of an Inhibitor of CDK5-Mediated PPARγ Phosphorylation.

Thiazolidinedione PPARγ agonists such as rosiglitazone and pioglitazone are effective antidiabetic drugs, but side effects have limited their use. It has been posited that their positive antidiabetic effects are mainly mediated by the inhibition of the CDK5-mediated Ser273 phosphorylation of PPARγ, whereas the side effects are linked to classical PPARγ agonism. Thus compounds that inhibit PPARγ Ser273 phosphorylation but lack classical PPARγ agonism have been sought as safer antidiabetic therapies. Herein we report the discovery by virtual screening of 10, which is a potent PPARγ binder and in vitro inhibitor of the CDK5-mediated phosphorylation of PPARγ Ser273 and displays negligible PPARγ agonism in a reporter gene assay. The pharmacokinetic properties of 10 are compatible with oral dosing, enabling preclinical in vivo testing, and a 7 day treatment demonstrated an improvement in insulin sensitivity in the ob/ob diabetic mouse model.

Measurement of unbound drug exposure in brain: modeling of pH partitioning explains diverging results between the brain slice and brain homogenate methods.

Currently used methodology for determining unbound drug exposure in brain combines measurement of the total drug concentration in the whole brain in vivo with estimation of brain tissue binding from one of two available in vitro methods: equilibrium dialysis of brain homogenate and the brain slice uptake method. This study of 56 compounds compares the fraction of unbound drug in brain (f(u,brain)), determined using the brain homogenate method, with the unbound volume of distribution in brain (V(u,brain)), determined using the brain slice method. Discrepancies were frequent and were primarily related to drug pH partitioning, attributable to the preservation of cellular structures in the slice that are absent in the homogenate. A mathematical model for pH partitioning into acidic intracellular compartments was derived to predict the slice V(u,brain) from measurements of f(u,brain) and drug pK(a). This model allowed prediction of V(u,brain) from f(u,brain) within a 2.2-fold error range for 95% of the drugs compared with a 4.5-fold error range using the brain homogenate f(u,brain) method alone. The greatest discrepancies between the methods occurred with compounds that are actively transported into brain cells, including gabapentin, metformin, and prototypic organic cation transporter substrates. It was concluded that intrabrain drug distribution is governed by several diverse mechanisms in addition to nonspecific binding and that the slice method is therefore more reliable than the homogenate method. As an alternative, predictions of V(u,brain) can be made from homogenate f(u,brain) using the pH partition model presented, although this model does not take into consideration possible active brain cell uptake.

Overlapping but distinct: Distal connectivity dissociates hand and tool processing networks.

The processes and organizational principles of information involved in object recognition have been a subject of intense debate. These research efforts led to the understanding that local computations and feedforward/feedback connections are essential to our representations and their organization. Recent data, however, has demonstrated that distal computations also play a role in how information is locally processed. Here we focus on how long-range connectivity and local functional organization of information are related, by exploring regions that show overlapping category-preferences for two categories and testing whether their connections are related with distal representations in a category-specific way. We used an approach that relates functional connectivity with distal areas to local voxel-wise category-preferences. Specifically, we focused on two areas that show an overlap in category-preferences for tools and hands-the inferior parietal lobule/anterior intraparietal sulcus (IPL/aIPS) and the posterior middle temporal gyrus/lateral occipital temporal cortex (pMTG/LOTC) - and how connectivity from these two areas relate to voxel-wise category-preferences in two ventral temporal regions dedicated to the processing of tools and hands separately-the left medial fusiform gyrus and the fusiform body area respectively-as well as across the brain. We show that the functional connections of the two overlap areas correlate with categorical preferences for each category independently. These results show that regions that process both tools and hands maintain object topography in a category-specific way. This potentially allows for a category-specific flow of information that is pertinent to computing object representations.

Decoding stimuli (tool-hand) and viewpoint invariant grasp-type information.

When we see a manipulable object (henceforth tool) or a hand performing a grasping movement, our brain is automatically tuned to how that tool can be grasped (i.e., its affordance) or what kind of grasp that hand is performing (e.g., a power or precision grasp). However, it remains unclear where visual information related to tools or hands are transformed into abstract grasp representations. We therefore investigated where different levels of abstractness in grasp information are processed: grasp information that is invariant to the kind of stimuli that elicits it (tool-hand invariance); and grasp information that is hand-specific but viewpoint-invariant (viewpoint invariance). We focused on brain areas activated when viewing both tools and hands, i.e., the posterior parietal cortices (PPC), ventral premotor cortices (PMv), and lateral occipitotemporal cortex/posterior middle temporal cortex (LOTC/pMTG). To test for invariant grasp representations, we presented participants with tool images and grasp videos (from first or third person perspective; 1pp or 3pp) inside an MRI scanner, and cross-decoded power versus precision grasps across (i) grasp perspectives (viewpoint invariance), (ii) tool images and grasp 1pp videos (tool-hand 1pp invariance), and (iii) tool images and grasp 3pp videos (tool-hand 3pp invariance). Tool-hand 1pp, but not tool-hand 3pp, invariant grasp information was found in left PPC, whereas viewpoint-invariant information was found bilaterally in PPC, left PMv, and left LOTC/pMTG. These findings suggest different levels of abstractness-where visual information is transformed into stimuli-invariant grasp representations/tool affordances in left PPC, and viewpoint invariant but hand-specific grasp representations in the hand network.

A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling.

Evaluation and optimization of physicochemical and metabolic properties of compounds are a crucial component of the drug development process. Continuous access to this information during the design-make-test-analysis cycle enables identification of chemical entities with suitable properties for efficient project progression. In this study, we describe an integrated and automated assay panel (DMPK Wave 1) that informs weekly on lipophilicity, solubility, human plasma protein binding, and metabolic stability in rat hepatocytes and human liver microsomes. All assays are running in 96-well format with ultraperformance liquid chromatography-mass spectrometry (MS)/MS as read-out. A streamlined overall workflow has been developed by optimizing all parts of the process, including shipping of compounds between sites, use of fit-for-purpose equipment and information systems, and technology for compound requesting, data analysis, and reporting. As a result, lead times can be achieved that well match project demands across sites independently of where compounds are synthesized. This robust screening strategy is run on a weekly basis and enables optimization of structure-activity relationships in parallel with DMPK properties to allow efficient and informed decision making.

Accelerated drug discovery by rapid candidate drug identification.

The eventual candidate drug (CD) is often already synthesized during early drug discovery but not nominated until much later. To facilitate the rapid identification of a potential CD, a thoroughly worked-out CD target profile (CDTP) with criteria acceptable for the disease target product profile (TPP) is required at the start of lead generation (LG). In addition to driving the compound property optimization, the preclinical project team has to understand the ultimate goal to be able to rapidly identify and progress a potential CD. A screening cascade with meaningful and well-balanced progression criteria based on the CDTP is required to rapidly filter out unwanted compounds and to progress a potential CD through the cascade to candidate selection.

High-throughput screening of drug-brain tissue binding and in silico prediction for assessment of central nervous system drug delivery.

A high-throughput method for rapid screening of in vitro drug-brain homogenate binding is presented. The method is based on a straightforward sample pooling approach combining equilibrium dialysis with liquid chromatography mass spectrometry (LCMS). A strong correlation of fraction unbound in brain (fu) between single compound measurements and 25-pooled compounds (R2 = 0.906) was obtained for a selection of structurally diverse CNS compounds with a wide range of fractions unbound. Effects of brain homogenate dilution and dialysis time were investigated. To the best of our knowledge, it was the first time that we have demonstrated consistent fraction unbound in mouse and rat brain homogenate, revealing the drug-tissue partitioning mechanism predominated by hydrophobic interaction. On the basis of this finding, a generic approach to estimate drug binding to various tissues is proposed. A robust and interpretable QSAR for fu prediction is also presented by statistical modeling.

Polarity-specific transcranial direct current stimulation effects on object-selective neural responses in the inferior parietal lobe.

Neuromodulation techniques such as transcranial direct current stimulation (tDCS) are routinely used for treating neurological and neuropsychiatric disorders, and for enhancement of cognitive abilities. Recently, their effectiveness in modulating behavioral and neural responses has been questioned. Here we use excitatory and inhibitory tDCS prior to a functional magnetic resonance imaging (fMRI) experiment to show that neural responses for an area's preferred stimuli depend on the polarity of stimulation. This is an important, yet overlooked, data point in demonstrating the effectiveness of these stimulation techniques. Our results show that response preferences in the target area are dependent on the polarity of the tDCS session preceding the fMRI experiment - these preferences are less distinct in the cathodal than in the anodal session. As such, we show unequivocally that tDCS modulates neural responses. This result is of the utmost importance in demonstrating the effectiveness of tDCS for clinical and experimental purposes.

Presentation of a structurally diverse and commercially available drug data set for correlation and benchmarking studies.

A multivariate analysis of drugs on the Swedish market was the basis for the selection of a small, physicochemically diverse set of 24 drug compounds. Factors such as structural diversity, commercial availability, price, and a suitable analytical technique for quantification were considered in the selection. Lipophilicity, pKa, solubility, and permeability across human Caco-2 cell monolayers were measured for the compiled data set. The results show that, by use of a physicochemically diverse data set, experimental responses over a wide range were obtained. The paper also shows how experimental difficulties due to the diversity of the data set can be overcome. We anticipate that this data set can serve as a benchmark set for validation of new experimental techniques or in silico models. It can also be used as a diverse starting data set for the development of new computational models.

Macrocyclic Prodrugs of a Selective Nonpeptidic Direct Thrombin Inhibitor Display High Permeability, Efficient Bioconversion but Low Bioavailability.

The only oral direct thrombin inhibitors that have reached the market, ximelagatran and dabigatran etexilat, are double prodrugs with low bioavailability in humans. We have evaluated an alternative strategy: the preparation of a nonpeptidic, polar direct thrombin inhibitor as a single, macrocyclic esterase-cleavable (acyloxy)alkoxy prodrug. Two homologous prodrugs were synthesized and displayed high solubilities and Caco-2 cell permeabilities, suggesting high absorption from the intestine. In addition, they were rapidly and completely converted to the active zwitterionic thrombin inhibitor in human hepatocytes. Unexpectedly, the most promising prodrug displayed only moderately higher oral bioavailability in rat than the polar direct thrombin inhibitor, most likely due to rapid metabolism in the intestine or the intestinal wall. To the best of our knowledge, this is the first in vivo ADME study of macrocyclic (acyloxy)alkoxy prodrugs, and it remains to be established if the modest increase in bioavailability is a general feature of this category of prodrugs or not.

Structural bases of the redox-dependent conformational switch in the serpin PAI-2.

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.

The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1' residues acting as a "bait" for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into beta-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor-donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in beta-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into beta-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.