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Nonnucleoside reverse transcriptase inhibitors (NNRTIs) target HIV-1 reverse transcriptase (RT) by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation., Synopsis HIV-1 encodes reverse transcriptase (RT), an enzyme that is essential for virus replication. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV-1 RT. In HIV-1-infected cells NNRTIs block the RT-catalyzed synthesis of a double-stranded DNA copy of the viral genomic RNA, which is an early step in the virus life cycle. Potent NNRTIs have the novel feature of promoting the interaction between the two RT subunits. However, the importance of this effect on the inhibition of HIV-1 replication has not been defined. In this study, the authors show that potent NNRTIs block an additional step in the virus life cycle. NNRTIs increase the intracellular processing of viral polyproteins called Gag and Gag-Pol that express the HIV-1 structural proteins and viral enzymes. Enhanced polyprotein processing is associated with a decrease in viral particles released from NNRTI-treated cells. NNRTI enhanced polyprotein processing is likely due to the drug binding to RT, expressed as part of the Gag-Pol polyprotein and promoting the interaction between separate Gag-Pol polyproteins. This leads to premature activation of the Gag-Pol embedded HIV-1 protease, resulting in a decrease in full-length viral polyproteins available for assembly and budding from the host cell membrane. This study provides proof-of-concept that small molecules can modulate the interactions between Gag-Pol polyproteins and suggests a new target for the development of HIV-1 antiviral drugs.

Severe human malaria is attributable to an excessive sequestration of Plasmodium falciparum–infected and uninfected erythrocytes in vital organs. Strains of P. falciparum that form rosettes and employ heparan sulfate as a host receptor are associated with development of severe forms of malaria. Heparin, which is similar to heparan sulfate in that it is composed of the same building blocks, was previously used in the treatment of severe malaria, but it was discontinued due to the occurrence of serious side effects such as intracranial bleedings. Here we report to have depolymerized heparin by periodate treatment to generate novel glycans (dGAG) that lack anticoagulant-activity. The dGAGs disrupt rosettes, inhibit merozoite invasion of erythrocytes and endothelial binding of P. falciparum–infected erythrocytes in vitro, and reduce sequestration in in vivo models of severe malaria. An intravenous injection of dGAGs blocks up to 80% of infected erythrocytes from binding in the micro-vasculature of the rat and releases already sequestered parasites into circulation. P. falciparum–infected human erythrocytes that sequester in the non-human primate Macaca fascicularis were similarly found to be released in to the circulation upon a single injection of 500 μg of dGAG. We suggest dGAGs to be promising candidates for adjunct therapy in severe malaria., Synopsis Severe Plasmodium falciparum malaria is common and in part the result of an excessive binding of infected erythrocytes in the microvasculature. The parasite employs heparan sulfate during the adherence to the vascular endothelium and to erythrocytes. Heparin, which is related to heparan sulfate in that it is composed of the same building blocks, was here periodate-treated to generate depolymerized glycosaminoglycans (dGAGs) that possess no anticoagulant activity. The dGAGs disrupt erythrocyte and endothelial binding of P. falciparum–infected erythrocytes in vitro. An intravenous injection of dGAGs blocks infected erythrocytes from binding in the micro-vasculature of the rat and releases already sequestrated parasites into circulation both in the rat and in a non-human primate. If this approach is successfully translated to the clinical setting, it may offer help to patients whereby the injection of a dGAG releases already sequestered parasite-infected erythrocytes and re-establishes the micro-vascular blood flow. The authors suggest dGAGs to be promising candidates of adjunct therapy that may have an important impact on malaria mortality.

G protein–coupled receptors (GPCRs) are one of the largest families of proteins, and here we scan the recently sequenced chicken genome for GPCRs. We use a homology-based approach, utilizing comparisons with all human GPCRs, to detect and verify chicken GPCRs from translated genomic alignments and Genscan predictions. We present 557 manually curated sequences for GPCRs from the chicken genome, of which 455 were previously not annotated. More than 60% of the chicken Genscan gene predictions with a human ortholog needed curation, which drastically changed the average percentage identity between the human–chicken orthologous pairs (from 56.3% to 72.9%). Of the non-olfactory chicken GPCRs, 79% had a one-to-one orthologous relationship to a human GPCR. The Frizzled, Secretin, and subgroups of the Rhodopsin families have high proportions of orthologous pairs, although the percentage of amino acid identity varies. Other groups show large differences, such as the Adhesion family and GPCRs that bind exogenous ligands. The chicken has only three bitter Taste 2 receptors, and it also lacks an ortholog to human TAS1R2 (one of three GPCRs in the human genome in the Taste 1 receptor family [TAS1R]), implying that the chicken's ability and mode of detecting both bitter and sweet taste may differ from the human's. The chicken genome contains at least 229 olfactory receptors, and the majority of these (218) originate from a chicken-specific expansion. To our knowledge, this dataset of chicken GPCRs is the largest curated dataset from a single gene family from a non-mammalian vertebrate. Both the updated human GPCR dataset, as well the chicken GPCR dataset, are available for download., Synopsis Man and chicken are very different, but how is that difference related to our respective gene repertoire? The authors studied the family of G protein–coupled receptors (GPCRs), which in man contains about 791 proteins. These are found in the cell membrane, where they recognize substances, thereby functioning as mediators of signals across the cellular membrane. GPCRs respond to physiologically important substances such as hormones and neurotransmitters. In this paper, the publicly available genomic sequence from the domestic chicken is used to identify the entire repertoire of GPCRs in this species. The authors found 557 GPCRs and compared the chicken and human receptors; they concluded that out of the 328 chicken receptors that are not involved in olfaction, more than 250 have a corresponding human receptor. The majority of the differences between the chicken and man are within three groups of GPCRs—the receptors for olfaction, bitter taste, and the receptors involved in the immune system. The chicken GPCR sequences obtained here will be useful for identification of GPCRs in other species that are more distantly related to man, such as fish or insects. The domestic chicken represents the leading experimental model among the avian species and also serves as an important source of food worldwide.

We have developed a software program that weights and integrates specific properties on the genes in a pathogen so that they may be ranked as drug targets. We applied this software to produce three prioritized drug target lists for Mycobacterium tuberculosis, the causative agent of tuberculosis, a disease for which a new drug is desperately needed. Each list is based on an individual criterion. The first list prioritizes metabolic drug targets by the uniqueness of their roles in the M. tuberculosis metabolome (“metabolic chokepoints”) and their similarity to known “druggable” protein classes (i.e., classes whose activity has previously been shown to be modulated by binding a small molecule). The second list prioritizes targets that would specifically impair M. tuberculosis, by weighting heavily those that are closely conserved within the Actinobacteria class but lack close homology to the host and gut flora. M. tuberculosis can survive asymptomatically in its host for many years by adapting to a dormant state referred to as “persistence.” The final list aims to prioritize potential targets involved in maintaining persistence in M. tuberculosis. The rankings of current, candidate, and proposed drug targets are highlighted with respect to these lists. Some features were found to be more accurate than others in prioritizing studied targets. It can also be shown that targets can be prioritized by using evolutionary programming to optimize the weights of each desired property. We demonstrate this approach in prioritizing persistence targets., Synopsis The search for drugs to prevent or treat infections remains an urgent focus in infectious disease research. A new software program has been developed by the authors of this article that can be used to rank genes as potential drug targets in pathogens. Traditional prioritization approaches to drug target identification, such as searching the literature and trying to mentally integrate varied criteria, can quickly become overwhelming for the drug discovery researcher. Alternatively, one can computationally integrate different criteria to create a ranking function that can help to identify targets. The authors demonstrate the applicability of this approach on the genome of Mycobacterium tuberculosis, the organism that causes tuberculosis (TB), a disease for which new drug treatments are especially needed because of emerging drug-resistant strains. The experiences gained from this work will be useful for both wet-lab and informatics scientists working in infectious disease research; first, it demonstrates that ample public data already exist on the M. tuberculosis genome that can be tuned effectively for prioritizing drug targets. Second, the output from numerous freely available bioinformatics tools can be pushed to achieve these goals. Third, the methodology can easily be extended to other pathogens of interest. Currently studied TB targets are also highlighted in terms of the authors' ranking system, which should be useful for researchers focusing on TB drug discovery.

Of the many important signaling events that take place on the surface of a mammalian cell, activation of signal transduction pathways via interactions of cell surface receptors is one of the most important. Evidence suggests that cell surface proteins are not as freely diffusible as implied by the classic fluid mosaic model and that their confinement to membrane domains is regulated. It is unknown whether these dynamic localization mechanisms function to enhance signal transduction activation rate or to minimize cross talk among pathways that share common intermediates. To determine which of these two possibilities is more likely, we derive an explicit equation for the rate at which cell surface membrane proteins interact based on a Brownian motion model in the presence of endocytosis and exocytosis. We find that in the absence of any diffusion constraints, cell surface protein interaction rate is extremely high relative to cytoplasmic protein interaction rate even in a large mammalian cell with a receptor abundance of a mere two hundred molecules. Since a larger number of downstream signaling events needs to take place, each occurring at a much slower rate than the initial activation via association of cell surface proteins, we conclude that the role of co-localization is most likely that of cross-talk reduction rather than coupling efficiency enhancement., Synopsis Cells coordinate activities with their neighboring cells by releasing and responding to chemical signals, such as hormones and growth factors. These extracellular signals often are transmitted to the intracellular regulatory machinery through associations of freely diffusing cell surface receptors whose random movement (Brownian motion) results from collisions with thermally agitated lipid molecules. Receptors on the cell surface signaling proteins are co-localized transiently on membrane patches, but how such spatial restriction contributes to signal transduction is unclear. Batada and colleagues set out to determine which is more likely to be the biophysical function of co-localization: increasing coupling efficiency of slowly diffusing membrane proteins or reducing cross talk along pathways with common intermediates. Using a mathematical model to describe Brownian motion–based association of receptors on a 2-D spherical surface, the authors show that the rate of signal transduction initiation is extremely fast compared with the cytoplasmic signal relay events, even when the number of receptors is low. Because enhancement of a process that is already fast is not beneficial to the cell, minimization of detrimental cross talk among pathways is the most likely reason why cells need to co-localize plasma membrane signaling proteins.

Artemisinins, derived from the wormwood herb Artemisia annua, are the most potent antimalarial drugs currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. Here we use yeast, Saccharamyces cerevisiae, to probe the core working mechanism of this class of antimalarial agents. We demonstrate that artemisinin's inhibitory effect is mediated by disrupting the normal function of mitochondria through depolarizing their membrane potential. Moreover, in a genetic study, we identify the electron transport chain as an important player in artemisinin's action: Deletion of NDE1 or NDI1, which encode mitochondrial NADH dehydrogenases, confers resistance to artemisinin, whereas overexpression of NDE1 or NDI1 dramatically increases sensitivity to artemisinin. Mutations or environmental conditions that affect electron transport also alter host's sensitivity to artemisinin. Sensitivity is partially restored when the Plasmodium falciparum NDI1 ortholog is expressed in yeast ndi1 strain. Finally, we showed that artemisinin's inhibitory effect is mediated by reactive oxygen species. Our results demonstrate that artemisinin's effect is primarily mediated through disruption of membrane potential by its interaction with the electron transport chain, resulting in dysfunctional mitochondria. We propose a dual role of mitochondria played during the action of artemisinin: the electron transport chain stimulates artemisinin's effect, most likely by activating it, and the mitochondria are subsequently damaged by the locally generated free radicals., Synopsis Malaria kills at least 1 million people worldwide a year. Recent years saw the rapid emergence of drug-resistant malaria strains. Artemisinins, derived from the Chinese wormwood herb Artemisia annua, are the most potent antimalarials currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. In this article, Li et al. investigated yeast as a model to probe the core working mechanism of this class of antimalarials. They showed that artemisinin can disrupt the normal function of mitochondria by depolarizing its membrane potential, and that artemisinin's effect can be affected by its interaction with the mitochondrial electron transport chain, an apparatus that couples oxygen oxidation and energy generation in the cell. They proposed a dual role of mitochondria played during the action of artemisinin: the electron transport chain likely activates artemisinin, and the mitochondria are subsequently damaged by the locally generated free radicals associated with this activation. The research has provided a fine tool for the study of the mechanism of artemisinin in a model organism (yeast), and laid the framework for a set of possible future experiments to be conducted in yeast and malaria parasites.

The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2−/− are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2+/− mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies., Synopsis The Ran-binding protein 2 (RanBP2) is a large protein with several domains. Although several protein partners were found to interact with selective domains of RanBP2, none to this date were found toward its large leucine-rich domain (LD). Cell-based experiments support several roles of RanBP2 in cell function, such as the production of functional proteins, control of protein trafficking between the nuclear and cytosol compartments, and control of multiple facets underlying cell division. Still, the genetic and physiological implications of the interactions between RanBP2 and its partners and of the function of RanBP2 in a whole-animal model remain elusive. The authors report the identification of two novel mitochondrial partners of the LD of RanBP2, Cox11 and hexokinase type I (HKI); and with multidisciplinary approaches probe the role of RanBP2 and its LD on Cox11, HKI, and functions allied to these. The authors found that RanBP2 exhibits chaperone activity toward HKI and Cox11. RanBP2 and Cox11 profoundly modulate HKI activity. Moreover, partial loss-of-function of RanBP2 in a mouse model induces deficits in growth rates and breakdown of glucose, promotes the down-regulation of HKI and ATP levels selectively in the central nervous system, and impairs visual function. These findings support a critical role of RanBP2 and its partners in metabolic processes and allied disease states.

The study of associations between two biomolecules is the key to understanding molecular function and recognition. Molecular function is often thought to be determined by underlying structures. Here, combining a single-molecule study of protein binding with an energy-landscape–inspired microscopic model, we found strong evidence that biomolecular recognition is determined by flexibilities in addition to structures. Our model is based on coarse-grained molecular dynamics on the residue level with the energy function biased toward the native binding structure (the Go model). With our model, the underlying free-energy landscape of the binding can be explored. There are two distinct conformational states at the free-energy minimum, one with partial folding of CBD itself and significant interface binding of CBD to Cdc42, and the other with native folding of CBD itself and native interface binding of CBD to Cdc42. This shows that the binding process proceeds with a significant interface binding of CBD with Cdc42 first, without a complete folding of CBD itself, and that binding and folding are then coupled to reach the native binding state. The single-molecule experimental finding of dynamic fluctuations among the loosely and closely bound conformational states can be identified with the theoretical, calculated free-energy minimum and explained quantitatively in the model as a result of binding associated with large conformational changes. The theoretical predictions identified certain key residues for binding that were consistent with mutational experiments. The combined study identified fundamental mechanisms and provided insights about designing and further exploring biomolecular recognition with large conformational changes., Synopsis Biomolecular function (e.g., binding) is often thought to be determined by the underlying molecular structure. There are more and more findings that molecular binding sometimes involves large conformational changes in various stages of cell function. Addressing this issue will answer the critical questions about how molecular function is determined by conformational flexibility and dynamics in addition to structure. Combining a single-molecule fluorescence study of flexible protein binding with an energy-landscape–inspired microscopic molecular dynamics model, the authors found strong evidence that biomolecular recognition is determined by flexibility and large conformational changes in addition to structure. The single-molecule study shows conformational fluctuations of the protein complex that involve bound and loosely bound states, which can be quantitatively explained in the authors' model as a result of cooperative binding. Theoretical predictions about the key residues are consistent with mutational experiments. Identifying the key residues for binding provides a structural basis for designing drugs that will target those critical residues.

A bold new effort to disrupt every gene in the mouse genome necessitates systematic, interdisciplinary approaches to analyzing patterning defects in the mouse embryo. We present a novel, rapid, and inexpensive method for obtaining high-resolution virtual histology for phenotypic assessment of mouse embryos. Using osmium tetroxide to differentially stain tissues followed by volumetric X-ray computed tomography to image whole embryos, isometric resolutions of 27 μm or 8 μm were achieved with scan times of 2 h or 12 h, respectively, using mid-gestation E9.5–E12.5 embryos. The datasets generated by this method are immediately amenable to state-of-the-art computational methods of organ patterning analysis. This technique to assess embryo anatomy represents a significant improvement in resolution, time, and expense for the quantitative, three-dimensional analysis of developmental patterning defects attributed to genetically engineered mutations and chemically induced embryotoxicity., Synopsis Developmental biology is entering the digital age, thanks to advancements in imaging technologies, instrumentation, and software. These advancements are converging with discoveries in developmental biology to deliver unprecedented insight into how human development is impacted by the products we use, the environment we live in, and our genetic composition. Industrial societies are becoming increasingly concerned with the exposure of women and their unborn fetuses to pharmaceuticals and commonplace household chemicals. In addition, understanding genetic causes of birth defects is now possible through the isolation of specific genes, which can be efficiently disrupted in embryos, and subsequently observed for birth defects. Such studies of embryotoxicity typically involve the use of mouse embryos. However, evaluation of mouse embryos in the past has involved expensive and cumbersome external inspection and thin sectioning for view under the microscope. As such, developmental biologists have eagerly anticipated the advent of tools that would allow them to routinely assess the complex and dynamic organization of embryos using techniques that are fast and inexpensive. In this article, the authors introduce a rapid, high-quality, and inexpensive technique for the three-dimensional visualization of mouse embryos using X-ray computed tomography that is ideally suited for researchers in pharmaceutical, industrial, and academic laboratories.

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation., Synopsis The authors have utilized Caenorhabditis elegans, and yeast genetics, combined with mammalian tissue and cell culture experiments to investigate the mechanism of action of a unique set of small molecules. These molecules are active in tissue models of urinary incontinence and allow for increased bladder filling. In the course of studying sensitivity and resistance to these compounds, Fitzgerald et al. uncovered novel alleles of RGS and Gq proteins. Further characterization of one such allele identified that its action conferred a hypo-adaptive phenotype on yeast during pheromone signaling assays. Their data as a whole indicate that these small molecules are able to diminish signaling from G-protein coupled receptors (GPCR) downstream of the receptors themselves. Since GPCR signaling is very important in many diseases in humans, the novel mechanism of these compounds may offer new ways to treat human disease.