Distinct Structural Classes Research Articles

Endophytic Streptomyces: an underexplored source with potential for novel natural drug discovery and development.

Streptomyces has long been considered as key sources for natural compounds discovery in medicine and agriculture. These compounds have been demonstrated to possess different biological activities, including antibiotic, antifungal, anticancer, and antiviral effects. As a result, new pharmaceuticals and antibiotics have been developed. Nevertheless, there have been only a few novel discoveries of bioactive compounds in the past decades from Streptomyces in natural habitats. There is, therefore, now a renewed search for new Streptomyces species having the potential to produce many compounds from one strain in lesser explored natural habitats that may be helpful in fighting diseases. Consequently, modern genome mining approaches are imperative for discovering structurally novel natural compounds with therapeutic applications from untapped sources. In light of these facts, endophytic Streptomyces from plants may offer new avenues for the discovery of bioactive compounds with distinctive chemical properties and activities. In the present review, we present the progress made in isolating natural compounds from endophytic Streptomyces originating from plants which have remarkable antimicrobial, cytotoxic, and antifungal properties. A different of distinct structural classes of compounds were reported from endophytic Streptomyces, such as indolosequiterpene, macrolides, flavones, peptides, naphthoquinones, and terpenoids. Further, we discussed modern genomics progress in finding biosynthetic gene clusters (BGCs) encoding compounds. Overall, this review might provide valuable insights into the potential for novel drug discovery from untapped endophytic Streptomyces in the future.

LVPocket: integrated 3D global-local information to protein binding pockets prediction with transfer learning of protein structure classification

BackgroundPrevious deep learning methods for predicting protein binding pockets mainly employed 3D convolution, yet an abundance of convolution operations may lead the model to excessively prioritize local information, thus overlooking global information. Moreover, it is essential for us to account for the influence of diverse protein folding structural classes. Because proteins classified differently structurally exhibit varying biological functions, whereas those within the same structural class share similar functional attributes.ResultsWe proposed LVPocket, a novel method that synergistically captures both local and global information of protein structure through the integration of Transformer encoders, which help the model achieve better performance in binding pockets prediction. And then we tailored prediction models for data of four distinct structural classes of proteins using the transfer learning. The four fine-tuned models were trained on the baseline LVPocket model which was trained on the sc-PDB dataset. LVPocket exhibits superior performance on three independent datasets compared to current state-of-the-art methods. Additionally, the fine-tuned model outperforms the baseline model in terms of performance.Scientific contributionWe present a novel model structure for predicting protein binding pockets that provides a solution for relying on extensive convolutional computation while neglecting global information about protein structures. Furthermore, we tackle the impact of different protein folding structures on binding pocket prediction tasks through the application of transfer learning methods.Graphical

Abstract 1433: FOXA1 alterations distinctively drive prostate tumorigenesis or therapy resistance in mice

Abstract Androgen receptor (AR) signaling is critical for survival of prostate cancer (PCa) cells, thus making androgen deprivation therapy (ADT; e.g., castration) as the mainstay for treatment. Notably, oncogenic functions of AR rely on chromatin-binding regulatory proteins, which includes a pioneer transcription factor called FOXA1. FOXA1 de-compacts chromatin to enable AR’s binding to the DNA and expression of it's target genes. Recently, our lab found FOXA1 alterations to recur within three distinct structural classes in over 35% of metastatic castration resistant PCa (mCRPC) in Caucasian men. Subsequent studies found FOXA1 mutations to be prevalent in over 40% of primary PCa in Chinese men, positioning FOXA1 as a principal oncogene in this disease. Yet, hitherto, no studies have defined the pathobiology of FOXA1 alterations in mouse models. Here, we have developed the first-in-field transgenic mice with conditional overexpression of FOXA1 mutants in the prostate luminal epithelia. We found truncal FOXA1 Class1 mutants to initiate luminal hyperplasia in a monogenic model, or hyperproliferative prostate adenocarcinoma in a compound Trp53-deficient genetic background. Mechanistically, Class1 mutants concurrently upregulate the AR and mTORC1/2 pathways to drive transformation, with this being the first report of FOXA1-driven PCa formation in mice. In contrast, FOXA1 Class2 mutants—which are acquired in mCRPC—do not drive prostate luminal transformation. Instead, single-cell multi-omics (RNA+ATAC) profiling of Class2-mutant mouse prostate tissue uncovered extensive transcriptional remodeling of epithelial cells into a luminal stem-like cell fate, leading to a dramatic 15-20-fold expansion of the progenitor population vs control tissue. These luminal stem cells are similar to the Club/Hillock cells detected in the human prostate that have been implicated in driving resistance to ADT. Consistently, we found Class2-mutant prostates to show minimal atrophy upon castration, with immunohistological assessment uncovering a higher density of Ki67+ luminal epithelial cells relative to wild type and Class1 tissues. Class2-mutant organoids also showed higher subcutaneous grafting ability in mice in limiting dilution assays. Mechanistically, we found the cistromically-dominant Class2 mutants to pioneer over 40,000 neo-enhancer elements harboring motifs of stemness-associated transcription factors, which in turn instruct the ADT-resistant luminal progenitor gene program. Altogether, findings from our mouse models uncover FOXA1’s versatility as a driver oncogene that, depending on the mutation type, either activates enhancer-wired luminal tumorigenesis (Class1-initiating event) or therapy resistance-associated stemness (Class2-promoting alteration) gene programs in the mouse prostate tissue. Citation Format: Sanjana Eyunni, Abhijit Parolia, Eleanor Young, James Matthew George, Rahul Mannan, Sandra E. Carson, Yuping Zhang, Jean Tien, Mustapha Jaber, Jie Luo, Matthew Pang, Rohit Mehra, Xuhong Cao, Fengyun Su, Rui Wang, Marcin Cieslik, Dong Kee Lee, Jianming Xu, Arul M. Chinnaiyan. FOXA1 alterations distinctively drive prostate tumorigenesis or therapy resistance in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1433.

Abstract 4336: Homologous recombination repair deficiency, and not recurrence, determines mutational burden and clonal dynamics in high grade serous ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy, killing more than 9,000 women each year in the United States. Nearly 80% of patients with HGSOC will experience recurrence within 5 years, but little is known about the mechanisms that drive this process. Up to 60% of the genome in an HGSOC tumor is impacted by structural variant mutations and pronounced intratumoral heterogeneity. Intratumor heterogeneity is believed to be a key feature of recurrence and therapy resistance with some evidence indicating diverse and complex mechanisms of the seeding of metastatic sites and recurrent tumors. We included 35 HGSOC patients for whom paired chemo-naive and chemoresistant tumors as well as germline DNA was available. Whole genome sequencing was used to identify somatic single nucleotide variants (SNV) insertion/deletion variants (indels), and structural variants (SVs) in each tumor. The number of somatic SVs and indels was higher in tumors with homologous recombination repair deficiency (HRD; P=0.000252 SVs, P=0.00385 Indel) but was not affected by recurrent status (P=0.826 SV, P=0.145 SNV/indel). Clonal composition and dynamics were measured using SNVs/indels as tumors progressed from chemo-naïve primary samples to recurrent chemo-resistant tumors. Surprisingly, few changes were observed in clonal abundance and complexity through progression. When this analysis was repeated with SVs homologous recombination repair proficient tumors tend to be polyclonal while HRD tumors tend to be monoclonal. Five denovo structural variant signatures were identified in our cohort and led to three distinct structural variant classes tumors defined by deletion, tumors defined by duplication, and tumors defined by copy number neutral changes (inversions, translocations and balanced complex events). Patients with tumors defined by deletions and copy number neutral changes carry pathogenic SVs correlating with reduced survival (P=0.00023), while DNA gains are less subject to pathogenic alterations. Each class displayed distinct regions of the chromosome that was frequently affected by large scale SV events (>5Mb), which was also observed in the HRP/HRD status. Although no regions were notably altered across recurrence, GO analysis revealed that recurrent tumors have a significantly reduced immune response, which was not seen in the primary tumors. Within a subset of tumors, we are plan to utilize Oxford Nanopore Technologies ultra long read sequencing which will allow for confirmation of SVs observed in the WGS as well as identification of more complex events that cannot be deciphered using WGS techniques. Citation Format: Michael A. Diaz, Nicole Gull, Pei-Chen Peng, Kate Lawrenson, Bobbie J. Rimel, Jenny Lester, Beth Karlan, Simon A. Gayther, Michelle R. Jones. Homologous recombination repair deficiency, and not recurrence, determines mutational burden and clonal dynamics in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4336.

Isochlorogenic Acid Glucosides from the Arabian Medicinal Plant Artemisia sieberi and Their Antimicrobial Activities.

A phytochemical investigation of the stems of the Arabian plant Artemisia sieberi afforded three new isochlorogenic acid derivatives, namely isochlorogenic acid A-3'-O-β-glucopyranoside (1), isochlorogenic acid A-3'-O-β-glucopyranoside methyl ester (2), and isochlorogenic acid C-3'-O-β-glucopyranoside (3), obtained along with thirteen known secondary metabolites belonging to distinct structural classes. The structures of the new metabolites were elucidated by modern spectroscopic techniues based on high-resolution mass spectrometry (HR-ESIMS) and 1D/2D nuclear magnetic resonance (NMR). All isolated compounds were tested for their potential antimicrobial activity against four different bacterial strains (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa), in addition to a fungal strain (Candida tropicalis), The results were expressed as the diameter of the clear zone (in millimetres) around each well. Compounds 1 and 3 (isochlorogenic acid A-3'-O-β-glucopyranoside and isochlorogenic acid C-3'-O-β-glucopyranoside, respectively) displayed remarkable antifungal effect and potent antibacterial activities against B. subtilis and S. aureus, respectively. 3α,4α-10β-trihydroxy-8α-acetyloxyguaian-12,6α-olide (6) and angelicoidenol 2-O-β-d-glucopyranoside (9) emerged as interesting dual antibacterial (selective on P. aeruginosa)/antifungal agents.

Structural and functional characterization of Mpp75Aa1.1, a putative beta-pore forming protein from Brevibacillus laterosporus active against the western corn rootworm.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (β-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.

M-Phenylenediammonium as a New Spacer for Dion-Jacobson Two-Dimensional Perovskites.

Two-dimensional (2D) halide perovskites have several distinct structural classes and exhibit great tunability, stability, and high potential for photovoltaic applications. Here, we report a new series of hybrid 2D perovskites in the Dion-Jacobson (DJ) class based on aromatic m-phenylenediammonium (mPDA) dications. The crystal structures of the DJ perovskite materials (mPDA)MAn-1PbnI3n+1 (n = 1-3) were solved and refined using single-crystal X-ray crystallography. The results indicate a short I···I interlayer distance of 4.00-4.04 Å for the (mPDA)MAn-1PbnI3n+1 (n = 2 and 3) structures, which is the shortest among DJ perovskites. However, Pb-I-Pb angles are as small as 158-160°, reflecting the large distortion of the inorganic framework, which results in larger band gaps for these materials than those in other DJ analogues. Density functional theory calculations suggest appreciable dispersion in the stacking direction, unlike the band structures of the Ruddlesden-Popper phases, which exhibit flat bands along the stacking direction. This is a consequence of the short interlayer I···I distances that can lead to interlayer electronic coupling across the layers. The solution-deposited films (nominal (mPDA)MAn-1PbnI3n+1 compositions of n = 1-6) reveal improved surface coverage with increasing nominal n value with the higher n films being composed of a mixture of n = 1 and bulk three-dimensional MAPbI3 perovskites. The films made from solutions of these materials behave differently from those of other 2D iodide perovskites, and their solar cells have a mixture of n = 1 DJ and MAPbI3 as light-absorbing semiconductors.

Indole scaffolds as a promising class of the aryl hydrocarbon receptor ligands.

The aryl hydrocarbon receptor (AhR), deemed initially as a xenobiotic sensor, plays multiple physiological roles and is involved in various pathophysiological processes and many diseases' etiology. Therefore, the therapeutic and chemopreventive targeting of AhR is a fundamental issue. To date, thousands of structurally diverse ligands of AhR have been identified. The bottleneck in targeting the AhR is that it is a Janus-faced player with beneficial vs. harmful effects in the ligand-specific context. A distinct structural class of the AhR ligands is those with indole-based scaffolds. The present review summarizes the knowledge on the existing indole-derived AhR ligands, comprising natural and dietary compounds, synthetic compounds including clinically used drugs, endogenous intermediary metabolites, and catabolites produced by human microbiota. The examples of novel, indole ring containing, rational design based AhR ligands are presented. The molecular, invitro, and invivo effects are described.

Bayesian inference assessment of protein secondary structure analysis using circular dichroism data - how much structural information is contained in protein circular dichroism spectra?

Circular dichroism spectroscopy is an important tool for determining the structural characteristics of biomolecules, particularly the secondary structure of proteins. In this paper we propose a Bayesian model that estimates the covariance structure within a measured spectrum and quantifies the uncertainty associated with the inferred secondary structures and characteristic spectra associated with each secondary structure type. Furthermore, we used tools from Bayesian model selection to determine the best secondary structure classification scheme and illustrate a technique for comparing whether or not two or more measured protein spectra share the same secondary structure. Our findings suggest that it is not possible to identify more than 3 distinct secondary structure classes from CD spectra above 175 nm. The inclusion of data from wavelengths between 175 and 200 nm did not substantially affect the ability to determine secondary structure fractions.

Re: Distinct Structural Classes of Activating FOXA1 Alterations in Advanced Prostate Cancer.

Re: Distinct Structural Classes of Activating FOXA1 Alterations in Advanced Prostate Cancer.

Mutations in FOXA1 Cluster into Distinct Structural and Phenotypic Classes

The effects of prostate cancer-associated mutations in FOXA1 depend on their position.

Reliable Target Prediction of Bioactive Molecules Based on Chemical Similarity Without Employing Statistical Methods.

The prediction of biological targets of bioactive molecules from machine-readable materials can be routinely performed by computational target prediction tools (CTPTs). However, the prediction of biological targets of bioactive molecules from non-digital materials (e.g., printed or handwritten documents) has not been possible due to the complex nature of bioactive molecules and impossibility of employing computations. Improving the target prediction accuracy is the most important challenge for computational target prediction. A minimum structure is identified for each group of neighbor molecules in the proposed method. Each group of neighbor molecules represents a distinct structural class of molecules with the same function in relation to the target. The minimum structure is employed as a query to search for molecules that perfectly satisfy the minimum structure of what is guessed crucial for the targeted activity. The proposed method is based on chemical similarity, but only molecules that perfectly satisfy the minimum structure are considered. Structurally related bioactive molecules found with the same minimum structure were considered as neighbor molecules of the query molecule. The known target of the neighbor molecule is used as a reference for predicting the target of the neighbor molecule with an unknown target. A lot of information is needed to identify the minimum structure, because it is necessary to know which part(s) of the bioactive molecule determines the precise target or targets responsible for the observed phenotype. Therefore, the predicted target based on the minimum structure without employing the statistical significance is considered as a reliable prediction. Since only molecules that perfectly (and not partly) satisfy the minimum structure are considered, the minimum structure can be used without similarity calculations in non-digital materials and with similarity calculations (perfect similarity) in machine-readable materials. Nine tools (PASS online, PPB, SEA, TargetHunter, PharmMapper, ChemProt, HitPick, SuperPred, and SPiDER), which can be used for computational target prediction, are compared with the proposed method for 550 target predictions. The proposed method, SEA, PPB, and PASS online, showed the best quality and quantity for the accurate predictions.

Abstract 4497: Distinct structural classes of activating FOXA1 alterations in prostate cancer progression

Abstract Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland. FOXA1 is frequently mutated in the hormone-receptor driven prostate, breast, bladder, and salivary gland tumors. In prostate luminal epithelial cells, wild-type FOXA1 delimits tissue-specific enhancers that are transcriptionally activated by AR, and extensively reprograms AR-activity in the transformed prostate epithelia. However, how FOXA1 alterations affect cancer development is unclear, with FOXA1 previously ascribed both tumor suppressive and oncogenic roles. In this study, we assemble an aggregate cohort of 1546 prostate cancers (PCa) and, for the first time, show that FOXA1 alterations fall into three distinct structural classes that diverge in clinical incidence, genetic co-alteration profiles, and oncogenic gain-of-functions. Notably, we find the three classes of FOXA1 alterations to collectively recur at a frequency of 35% in metastatic PCa. Class1 activating mutations originate in early PCa without concurrent ETS or SPOP alterations, selectively recur within the Wing2-region of the DNA-binding Forkhead domain (FKHD), confer enhanced chromatin mobility and binding frequency, and strongly trans-activate a luminal androgen receptor (AR) program of prostate oncogenesis. By contrast, class2 activating mutations are acquired in metastatic PCa, truncate the C-terminal regulatory domain of FOXA1, confer chromatin-binding dominance by increasing DNA affinity, and aberrantly activate the WNT/β-Catenin pathway to enable PCa metastasis. Finally, class3 genomic rearrangements are comprised of tandem duplications and translocations within the highly-syntenic FOXA1 locus. These structural variations amplify or reposition a conserved enhancer element, which we named FOXA1 Mastermind (FOXMIND), to drive overexpression of FOXA1 or other oncogenes, respectively. In summary, our study reaffirms the central role of FOXA1 in mediating AR-driven oncogenesis, and provides mechanistic insights into how different classes of FOXA1 alterations uniquely promote PCa initiation and/or metastatic progression. Furthermore, these results have direct implications in understanding the biology of other hormone-receptor driven cancers where similar FOXA1 alterations are found, and rationalize therapeutic co-targeting of oncogenic FOXA1-activity to extort more potent and durable disease remissions. Citation Format: Abhijit Parolia, Marcin Cieslik, Shih-Chun Chu, Lanbo Xiao, Takahiro Ouchi, Yuping Zhang, Xiaoju Wang, Pankaj Vats, Xuhong Cao, Fengyun Su, Rui Wang, Felix Feng, Yi-Mi Wu, Robert Lonigro, Dan R. Robinson, Arul M. Chinnaiyan. Distinct structural classes of activating FOXA1 alterations in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4497.

Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer.

SummaryForkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland1,2. FOXA1 is frequently mutated in the hormone-receptor driven prostate, breast, bladder, and salivary gland tumors3–8. However, how FOXA1 alterations affect cancer development is unclear, with FOXA1 previously ascribed both tumor suppressive9–11 and oncogenic12–14 roles. Here we assemble an aggregate cohort of 1546 prostate cancers (PCa) and show that FOXA1 alterations fall into three distinct structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class1 activating mutations originate in early PCa without ETS/SPOP alterations, selectively recur within the Wing2-region of the DNA-binding Forkhead domain (FKHD), enable enhanced chromatin mobility and binding frequency, and strongly transactivate a luminal androgen receptor (AR) program of prostate oncogenesis. By contrast, class2 activating mutations are acquired in metastatic PCa, truncate the C-terminal domain of FOXA1, enable dominant chromatin binding by increasing DNA affinity, and through TLE3 inactivation promote WNT-pathway driven metastasis. Finally, class3 genomic rearrangements are enriched in metastatic PCa, comprise of duplications and translocations within the FOXA1 locus, and structurally reposition a conserved regulatory element, herein denoted FOXA1 Mastermind (FOXMIND), to drive overexpression of FOXA1 or other oncogenes. Our study reaffirms the central role of FOXA1 in mediating AR-driven oncogenesis, and provides mechanistic insights into how different classes of FOXA1 alterations uniquely promote PCa initiation and/or metastatic progression. Furthermore, these results have direct implications in understanding the pathobiology of other hormone-receptor driven cancers and rationalize therapeutic co-targeting of FOXA1 activity.

The Melting Diagram of Protein Solutions and Its Thermodynamic Interpretation.

Here we present a novel method for the characterization of the hydration of protein solutions based on measuring and evaluating two-component wide-line 1H NMR signals. We also provide a description of key elements of the procedure conceived for the thermodynamic interpretation of such results. These interdependent experimental and theoretical treatments provide direct experimental insight into the potential energy surface of proteins. The utility of our approach is demonstrated through the examples of two proteins of distinct structural classes: the globular, structured ubiquitin; and the intrinsically disordered ERD10 (early response to dehydration 10). We provide a detailed analysis and interpretation of data recorded earlier by cooling and slowly warming the protein solutions through thermal equilibrium states. We introduce and use order parameters that can be thus derived to characterize the distribution of potential energy barriers inhibiting the movement of water molecules bound to the surface of the protein. Our results enable a quantitative description of the ratio of ordered and disordered parts of proteins, and of the energy relations of protein–water bonds in aqueous solutions of the proteins.

Quantifying structural dynamic heterogeneity in a dense two-dimensional equilibrium liquid.

We investigate local structural fluctuations in a model equilibrium fluid with the aim of better understanding the structural basis of locally heterogeneous dynamics identified in recent simulations and experimental studies of glass-forming liquids and other strongly interacting particle systems, such as lipid membranes, dusty plasmas, interfacial dynamics of crystals, the internal dynamics of proteins, etc. In particular, we utilize molecular dynamics simulation methods to study a single component Lennard-Jones condensed material at constant temperature in two dimensions over a range of densities covering both liquid and crystalline phase regimes. We identify three distinct structural classes of particles by examining the immediate neighborhood of individual particles relying on a solid-angle based tessellation technique. The area distribution of the neighborhoods reveals cages having hexagonal, pentagonal, and square symmetries. Pentagonal cells appear to be the predominant motif in the liquid phase, while the solid phase is dominated by hexagonal cells, as in the case of a perfect crystal. An examination of the spatial organization of particles belonging to each structural class further indicates that finite-size clusters of the hexagonal and pentagonal particle populations arise within both liquids and solids, and the size of these clusters grows in a complementary way as a function of density. Both particle populations form percolation clusters in the liquid-crystal coexistence regime. Interestingly, the populations of particles with different local structures, defined by the arrangement of neighboring particles, are found to maintain different diffusivities, as computed from the velocity autocorrelation function for each type of particle for all densities studied. Our analysis provides a new conceptual framework for understanding the structural origin of dynamical heterogeneity in soft materials.

Family-wide analysis of aminoacyl-sulfamoyl-3-deazaadenosine analogues as inhibitors of aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that precisely attach an amino acid to its cognate tRNA. This process, which is essential for protein translation, is considered a viable target for the development of novel antimicrobial agents, provided species selective inhibitors can be identified. Aminoacyl-sulfamoyl adenosines (aaSAs) are potent orthologue specific aaRS inhibitors that demonstrate nanomolar affinities in vitro but have limited uptake. Following up on our previous work on substitution of the base moiety, we evaluated the effect of the N3-position of the adenine by synthesizing the corresponding 3-deazaadenosine analogues (aaS3DAs). A typical organism has 20 different aaRS, which can be split into two distinct structural classes. We therefore coupled six different amino acids, equally targeting the two enzyme classes, via the sulfamate bridge to 3-deazaadenosine. Upon evaluation of the inhibitory potency of the obtained analogues, a clear class bias was noticed, with loss of activity for the aaS3DA analogues targeting class II enzymes when compared to the equivalent aaSA. Evaluation of the available crystallographic structures point to the presence of a conserved water molecule which could have importance for base recognition within class II enzymes, a property that can be explored in future drug design efforts.

A DNA structural alphabet provides new insight into DNA flexibility

DNA is a structurally plastic molecule, and its biological function is enabled by adaptation to its binding partners. To identify the DNA structural polymorphisms that are possible in such adaptations, the dinucleotide structures of 60 000 DNA steps from sequentially nonredundant crystal structures were classified and an automated protocol assigning 44 distinct structural (conformational) classes called NtC (for Nucleotide Conformers) was developed. To further facilitate understanding of the DNA structure, the NtC were assembled into the DNA structural alphabet CANA (Conformational Alphabet of Nucleic Acids) and the projection of CANA onto the graphical representation of the molecular structure was proposed. The NtC classification was used to define a validation score called confal, which quantifies the conformity between an analyzed structure and the geometries of NtC. NtC and CANA assignment were applied to analyze the structural properties of typical DNA structures such as Dickerson-Drew dodecamers, guanine quadruplexes and structural models based on fibre diffraction. NtC, CANA and confal assignment, which is accessible at the website https://dnatco.org, allows the quantitative assessment and validation of DNA structures and their subsequent analysis by means of pseudo-sequence alignment. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Acta_Cryst_D:2.

Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability

Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC50 values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical.

SMiLE-seq identifies binding motifs of single and dimeric transcription factors.

Resolving the DNA-binding specificities of transcription factors (TFs) is of critical value for understanding gene regulation. Here, we present a novel, semiautomated protein-DNA interaction characterization technology, selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq). SMiLE-seq is neither limited by DNA bait length nor biased toward strong affinity binders; it probes the DNA-binding properties of TFs over a wide affinity range in a fast and cost-effective fashion. We validated SMiLE-seq by analyzing 58 full-length human, mouse, and Drosophila TFs from distinct structural classes. All tested TFs yielded DNA-binding models with predictive power comparable to or greater than that of other in vitro assays. De novo motif discovery on all JUN-FOS heterodimers and several nuclear receptor-TF complexes provided novel insights into partner-specific heterodimer DNA-binding preferences. We also successfully analyzed the DNA-binding properties of uncharacterized human C2H2 zinc-finger proteins and validated several using ChIP-exo.