Molecular mechanisms and design principles for promiscuous inhibitors to avoid drug resistance: Lessons learned from HIV‐1 protease inhibition

Molecular recognition is central to biology and ranges from highly selective to broadly promiscuous. The ability to modulate specificity at will is particularly important for drug development, and discovery of mechanisms contributing to binding specificity is crucial for our basic understanding of biology and for applications in health care. In this study, we used computational molecular design to create a large dataset of diverse small molecules with a range of binding specificities. We then performed structural, energetic, and statistical analysis on the dataset to study molecular mechanisms of achieving specificity goals. The work was done in the context of HIV‐1 protease inhibition and the molecular designs targeted a panel of wild‐type and drug‐resistant mutant HIV‐1 protease structures. The analysis focused on mechanisms for promiscuous binding to bind robustly even to resistance mutants. Broadly binding inhibitors tended to be smaller in size, more flexible in chemical structure, and more hydrophobic in nature compared to highly selective ones. Furthermore, structural and energetic analyses illustrated mechanisms by which flexible inhibitors achieved binding; we found ligand conformational adaptation near mutation sites and structural plasticity in targets through torsional flips of asymmetric functional groups to form alternative, compensatory packing interactions or hydrogen bonds. As no inhibitor bound to all variants, we designed small cocktails of inhibitors to do so and discovered that they often jointly covered the target set through mechanistic complementarity. Furthermore, using structural plasticity observed in experiments, and potentially in simulations, is suggested to be a viable means of designing adaptive inhibitors that are promiscuous binders. Proteins 2015; 83:351–372. © 2014 Wiley Periodicals, Inc.     

Taxonomic,functional, and phylogenetic dimensions of rodent biodiversity along an extensive tropical elevational gradient

Relationships among taxonomic, functional, and phylogenetic dimensions of biodiversity provide insight about the relative contributions of ecological and evolutionary processes in structuring local assemblages. We used data for rodent species distributions from an extensive tropical elevational gradient to 1) describe elevational gradients for each of three dimensions of biodiversity, 2) evaluate the sufficiency of species richness as a surrogate for other dimensions, and 3) quantify the relative support for mechanisms that increase or decrease phylogenetic or functional dispersion. Taxonomic biodiversity was quantified by species richness, as well as by richness, evenness, diversity, dominance, and rarity at generic and familial levels. Morphological and categorical traits were used to estimate functional biodiversity, and an ultrametric mammalian supertree was used as the basis for estimating phylogenetic biodiversity. Elevational gradients of each dimension of biodiversity were strong, with significant linear and non‐linear components based on orthogonal polynomial regression. Empirical linear and non‐linear regression components were consistently different than those expected based on species richness for generic, familial, and phylogenetic biodiversity, but not for functional biodiversity. Nevertheless, the congruence of dimensions of biodiversity based on correlation analyses indicated that any one dimension is a useful surrogate for the other dimensions for rodents at Manu. Given variation in species richness, assemblages from lowland rainforests comprised more biodiversity than expected, whereas assemblages from cloud and elfin forests represented less biodiversity than expected. Warm temperatures, vertical complexity of the vegetation, and high productivity likely facilitate niche differentiation in rainforests, whereas cricetid rodents are competitively superior to other clades in the less structurally complex, less productive, and colder, high elevation habitats.     

MHC variation reflects the bottleneck histories of New Zealand passerines

Most empirical evidence suggests that balancing selection does not counter the effects of genetic drift in shaping postbottleneck major histocompatibility complex (MHC) genetic diversity when population declines are severe or prolonged. However, few studies have been able to include data from historical specimens, or to compare populations/species with different bottleneck histories. In this study, we examined MHC class II B and microsatellite diversity in four New Zealand passerine (songbird) species that experienced moderate to very severe declines. We compared diversity from historical samples (collected c. 1884–1938) to present‐day populations. Using a Bayesian framework, we found that the change in genetic diversity from historical to contemporary samples was affected by three main factors: (i) whether the data were based on MHC or microsatellite markers, (ii) species (as a surrogate for bottleneck severity) and (iii) whether the comparison between historical and contemporary samples was made using historical samples originating from the mainland, or using historical samples originating from islands. The greatest losses in genetic diversity occurred for the most severely bottlenecked species, particularly between historical mainland and contemporary samples. Additionally, where loss of diversity occurred, the change was greater for MHC genes compared to microsatellite loci.     

When and how did the terrestrial mid-Permian mass extinction occur? Evidence from the tetrapod record of the Karoo Basin,South Africa

A mid-Permian (Guadalupian epoch) extinction event at approximately 260 Ma has been mooted for two decades. This is based primarily on invertebrate biostratigraphy of Guadalupian–Lopingian marine carbonate platforms in southern China, which are temporally constrained by correlation to the associated Emeishan Large Igneous Province (LIP). Despite attempts to identify a similar biodiversity crisis in the terrestrial realm, the low resolution of mid-Permian tetrapod biostratigraphy and a lack of robust geochronological constraints have until now hampered both the correlation and quantification of terrestrial extinctions. Here we present an extensive compilation of tetrapod-stratigraphic data analysed by the constrained optimization (CONOP) algorithm that reveals a significant extinction event among tetrapods within the lower Beaufort Group of the Karoo Basin, South Africa, in the latest Capitanian. Our fossil dataset reveals a 74–80% loss of generic richness between the upper Tapinocephalus Assemblage Zone (AZ) and the mid-Pristerognathus AZ that is temporally constrained by a U–Pb zircon date (CA-TIMS method) of 260.259 ± 0.081 Ma from a tuff near the top of the Tapinocephalus AZ. This strengthens the biochronology of the Permian Beaufort Group and supports the existence of a mid-Permian mass extinction event on land near the end of the Guadalupian. Our results permit a temporal association between the extinction of dinocephalian therapsids and the LIP volcanism at Emeishan, as well as the marine end-Guadalupian extinctions.     

15-Hydroxyprostaglandin Dehydrogenase Generation of Electrophilic Lipid Signaling Mediators from Hydroxy Ω-3 Fatty Acids

15-Hydroxyprostaglandin dehydrogenase (15PGDH) is the primary enzyme catalyzing the conversion of hydroxylated arachidonic acid species to their corresponding oxidized metabolites. The oxidation of hydroxylated fatty acids, such as the conversion of prostaglandin (PG) E₂ to 15-ketoPGE₂, by 15PGDH is viewed to inactivate signaling responses. In contrast, the typically electrophilic products can also induce anti-inflammatory and anti-proliferative responses. This study determined that hydroxylated docosahexaenoic acid metabolites (HDoHEs) are substrates for 15PGDH. Examination of 15PGDH substrate specificity was conducted in cell culture (A549 and primary human airway epithelia and alveolar macrophages) using chemical inhibition and shRNA knockdown of 15PGDH. Substrate specificity is broad and relies on the carbon position of the acyl chain hydroxyl group. 14-HDoHE was determined to be the optimal DHA substrate for 15PGDH, resulting in the formation of its electrophilic metabolite, 14-oxoDHA. Consistent with this, 14-HDoHE was detected in bronchoalveolar lavage cells of mild to moderate asthmatics, and the exogenous addition of 14-oxoDHA to primary alveolar macrophages inhibited LPS-induced proinflammatory cytokine mRNA expression. These data reveal that 15PGDH-derived DHA metabolites are biologically active and can contribute to the salutary signaling actions of Ω-3 fatty acids.     

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca²⁺-ATPase (PMCA), which is critical for maintaining a low intracellular Ca²⁺ concentration ([Ca²⁺]_i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca²⁺]_i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca²⁺]_i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca²⁺]_i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.     

KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly

During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere–associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(−) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(−) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore–microtubule (k–MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(−) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k–MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly.     

The Impact of Recombination Hotspots on Genome Evolution of a Fungal Plant Pathogen

Recombination has an impact on genome evolution by maintaining chromosomal integrity, affecting the efficacy of selection, and increasing genetic variability in populations. Recombination rates are a key determinant of the coevolutionary dynamics between hosts and their pathogens. Historic recombination events created devastating new pathogens, but the impact of ongoing recombination in sexual pathogens is poorly understood. Many fungal pathogens of plants undergo regular sexual cycles, and sex is considered to be a major factor contributing to virulence. We generated a recombination map at kilobase-scale resolution for the haploid plant pathogenic fungus Zymoseptoria tritici. To account for intraspecific variation in recombination rates, we constructed genetic maps from two independent crosses. We localized a total of 10,287 crossover events in 441 progeny and found that recombination rates were highly heterogeneous within and among chromosomes. Recombination rates on large chromosomes were inversely correlated with chromosome length. Short accessory chromosomes often lacked evidence for crossovers between parental chromosomes. Recombination was concentrated in narrow hotspots that were preferentially located close to telomeres. Hotspots were only partially conserved between the two crosses, suggesting that hotspots are short-lived and may vary according to genomic background. Genes located in hotspot regions were enriched in genes encoding secreted proteins. Population resequencing showed that chromosomal regions with high recombination rates were strongly correlated with regions of low linkage disequilibrium. Hence, genes in pathogen recombination hotspots are likely to evolve faster in natural populations and may represent a greater threat to the host.