How does a small molecule bind at a cryptic binding site?

Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are “cryptic”: When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)—which performs its function by forming a PPI with its receptor—without incorporating any prior structural information about the ligands’ binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein–small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2–small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites.     

Different mechanisms influencing permeation of PDGF-AA and PDGF-BB across the blood-brain barrier

Platelet-derived growth factor (PDGF) exerts neurotrophic and neuromodulatory effects on the CNS. To determine the permeability of the blood-brain barrier (BBB) to PDGF, we examined the blood-to-brain influx of radioactively labeled PDGF isoforms (PDGF-AA and PDGF-BB) by multiple-time regression analysis after intravenous (i.v.) injection and by in-situ perfusion, and also determined the physicochemical characteristics which affect their permeation across the BBB, including lipophilicity (measured by octanol:buffer partition coefficient), hydrogen bonding (measured by differences in octanol : buffer and isooctane : buffer partition coefficients), serum protein binding (measured by capillary electrophoresis), and stability of PDGF in blood 10 min after i.v. injection (measured by HPLC). After i.v. bolus injection, neither 125I-PDGF-AA nor 125I-PDGF-BB crossed the BBB, their influx rates being similar to that of the vascular marker 99mTc-albumin. 125I-PDGF-AA degraded significantly faster in blood than 125I-PDGF-BB. PDGF-BB, however, was completely bound to a large protein in serum whereas PDGF-AA showed no binding. Thus, degradation might explain the poor blood-to-brain influx of PDGF-AA, whereas protein binding could explain the poor influx of circulating PDGF-BB. Despite their lack of permeation in the intact mouse, both 125I-PDGF-AA and 125I-PDGF-BB entered the brain by perfusion in blood-free buffer, and the significantly faster rate of 125I-PDGF-AA than 125I-PDGF-BB may be explained by the lower hydrogen bonding potential of 125I-PDGF-AA. Thus, the lack of significant distribution of PDGF from blood to brain is not because of the intrinsic barrier function of the BBB but probably because of degradation and protein binding. Information from these studies could be useful in the design of analogues for delivery of PDGF as a therapeutic agent.     

Antibodies to beta-amyloid decrease the blood-to-brain transfer of beta-amyloid peptide

Amyloid-beta peptides (Abeta) play an important role in the pathophysiology of dementia of the Alzheimer's type and in amyloid angiopathy. Abeta outside the CNS could contribute to plaque formation in the brain where its entry would involve interactions with the blood-brain barrier (BBB). Effective antibodies to Abeta have been developed in an effort to vaccinate against Alzheimer's disease. These antibodies could interact with Abeta in the peripheral blood, block the passage of Abeta across the BBB, or prevent Abeta deposition within the CNS. To determine whether the blocking antibodies act at the BBB level, we examined the influx of radiolabeled Abeta (125I-Abeta(1-40)) into the brain after ex-vivo incubation with the antibodies. Antibody mAb3D6 (élan Company) reduced the blood-to-brain influx of Abeta after iv bolus injection. It also significantly decreased the accumulation of Abeta in brain parenchyma. To confirm the in-vivo study and examine the specificity of mAb3D6, in-situ brain perfusion in serum-free buffer was performed after incubation of 125I-Abeta(1-40) with another antibody mAbmc1 (DAKO Company). The presence of mAbmc1 also caused significant reduction of the influx of Abeta into the brain after perfusion. Therefore, effective antibodies to Abeta can reduce the influx of Abeta(1-40) into the brain.     

Endogenous opiates: 1997

This paper is the twentieth installment of our annual review of research concerning the opiate system. It summarizes papers published during 1997 that studied the behavioral effects of the opiate peptides and antagonists, excluding the purely analgesic effects, although stress-induced analgesia is included. The specific topics covered this year include stress; tolerance and dependence; eating and drinking; alcohol; gastrointestinal, renal, and hepatic function; mental illness and mood; learning, memory, and reward; cardiovascular responses; respiration and thermoregulation; seizures and other neurologic disorders; electrical-related activity; general activity and locomotion; sex, pregnancy, and development; immunologic responses; and other behaviors.     

Assessing conservation priorities of xenarthrans in Argentina

In this study, we combine species distribution models with a reserve selection approach to assess the degree of representation of xenarthrans in the existing protected area network of Argentina, and to identify conservation priority areas that may help expand the current system. Species distribution models were developed from species’ occurrence records using a maximum entropy algorithm. Maps of species distributions were produced for 15 of the 16 species currently present in the country. To assess the performance of the existing protected area network in representing all modeled species, and to identify priority areas to expand the current reserve system, we used the software Zonation. Overall, all species modeled are represented in the existing protected area network. However, the percentage of their ranges covered by protected areas is very low (average = 6.7%; range = 1.7–17.6%). To represent at least 5% of the distribution of each species, 8.8% of the country’s area would be needed, and species with restricted ranges have the greatest increase in representation in this scenario. When 10% of the country is set aside for conservation, species representation increases considerably, again favoring range-restricted species. Most of the areas identified as conservation priorities are under strong anthropogenic pressures, including deforestation, agricultural expansion, and hunting. Our analysis provides a preliminary assessment of conservation priorities for the xenarthrans of Argentina, and we hope will serve as guideline to focus future conservation assessments at more refined scales.     

Neuregulin-1-beta1 enters brain and spinal cord by receptor-mediated transport

Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.     

Soluble receptor inhibits leptin transport

Evidence both from mice and cultured cells suggests an important role of soluble leptin receptors in obesity and leptin signaling. However, the direct effects of soluble receptors on leptin uptake by cells are not clear. This study shows that soluble leptin receptors antagonize the permeation of leptin across the mouse blood-brain barrier by reducing the binding and endocytosis of leptin. This is illustrated by analysis of radioactively labeled and fluorescent-tagged leptin in normal mice and in cultured cells overexpressing various forms of leptin receptors. Three constructs of soluble leptin receptors were generated in this study: ObRe (805 aa), ObR839, and ObR852. (125)I-leptin was injected intravenously and its influx rate from blood to brain determined by multiple-time regression analysis. Pre-incubation with ObR839 caused a significant reduction of leptin influx across the blood-brain barrier. Endocytosis assays and fluorescent image analysis further showed that ObRe, ObR839, and ObR852 failed to mediate leptin internalization and trafficking within the cells. Instead, these soluble receptors inhibited surface binding and endocytosis of leptin. Thus, we provide novel direct evidence both in vivo and in vitro that soluble receptors of leptin serve as antagonists of the transport of leptin.     

Effects of peptides on animal and human behavior: a review of studies published in the first twenty years of the journal Peptides

This review catalogs effects of peptides on various aspects of animal and human behavior as published in the journal Peptides in its first twenty years. Topics covered include: activity levels, addiction behavior, ingestive behaviors, learning and memory-based behaviors, nociceptive behaviors, social and sexual behavior, and stereotyped and other behaviors. There are separate tables for these behaviors and a short introduction for each section.