Phosphorylation-inducing chimera rewires oncogenic kinase to trigger apoptosis.

Hyperactive enzymes drive the pathology of several diseases, and classically, "occupancy-driven" drugs (e.g., active site or allosteric inhibitors) are used to target these enzymes.

Ultrasmall chemogenetic tags with group-transfer ligands.

Chemogenetic tags are valuable tools for studying functions of a given protein-of-interest (POI) lacking small-molecule ligands, but most tags are too large for several POIs. Here, we report two ultrasmall chemogenetic tags (mgTag and cTag) of 36 and 50 amino acids (aa) that, to the best of our knowledge, are the smallest reported. These tags exhibit -type reactivity with their ligands to append any moiety of interest to the tag.

Proteolysis-targeting chimeras with reduced off-targets.

Proteolysis-targeting chimeras (PROTACs) are molecules that induce proximity between target proteins and E3 ligases triggering target protein degradation. Pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other proteins, including zinc-finger (ZF) proteins, with vital roles in health and disease. This off-target degradation hampers the therapeutic applicability of pomalidomide-based PROTACs, requiring development of PROTAC design rules that minimize off-target degradation.

Development and Applications of Chimera Platforms for Tyrosine Phosphorylation.

Chimeric small molecules that induce post-translational modification (PTM) on a target protein by bringing it into proximity to a PTM-inducing enzyme are furnishing novel modalities to perturb protein function. Despite recent advances, such molecules are unavailable for a critical PTM, tyrosine phosphorylation. Furthermore, the contemporary design paradigm of chimeric molecules, formed by joining a noninhibitory binder of the PTM-inducing enzyme with the binder of the target protein, prohibits the recruitment of most PTM-inducing enzymes as their noninhibitory binders are unavailable.

Proximity-inducing modalities: the past, present, and future.

Living systems use proximity to regulate biochemical processes. Inspired by this phenomenon, bifunctional modalities that induce proximity have been developed to redirect cellular processes. An emerging example of this class is molecules that induce ubiquitin-dependent proteasomal degradation of a protein of interest, and their initial development sparked a flurry of discovery for other bifunctional modalities.

Functional characterization of a hypothetical protein (TTHA1873) from Thermus thermophilus.

Thermus thermophilus is an extremely thermophilic organism that thrives at a temperature of 65°C. T. thermophilus genome has ~2218 genes, out of which 66% (1482 genes) have been annotated, and the remaining 34% (736 genes) are assigned as hypothetical proteins.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps.

Structure of the hypothetical protein TTHA1873 from Thermus thermophilus.

The crystal structure of an uncharacterized hypothetical protein, TTHA1873 from Thermus thermophilus, has been determined by X-ray crystallography to a resolution of 1.78 Å using the single-wavelength anomalous dispersion method. The protein crystallized as a dimer in two space groups: P422 and P622.

Synthetic Reprogramming of Kinases Expands Cellular Activities of Proteins.

Phosphorylation-inducing chimeric small molecules (PHICS) can enable a kinase to act at a new cellular location or phosphorylate non-native substrates (neo-substrates)/ sites (neo-phosphorylations). We report a modular design and high-yielding synthesis of such PHICS that endowed multiple new activities to protein kinase C (PKC). For example, while PKC is unable to downregulate the activity of a gain-of-function variant (S180A) of Bruton's tyrosine kinase that evokes B cell malignancy phenotype, PHICS enabled PKC to induce inhibitory neo-phosphorylations on this variant.

Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes.

Prolonged Cas9 activity can hinder genome engineering as it causes off-target effects, genotoxicity, heterogeneous genome-editing outcomes, immunogenicity, and mosaicism in embryonic editing-issues which could be addressed by controlling the longevity of Cas9. Though some temporal controls of Cas9 activity have been developed, only cumbersome systems exist for modifying the lifetime. Here, we have developed a chemogenetic system that brings Cas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of Cas9 by the proteasome.

Phosphorylation-Inducing Chimeric Small Molecules.

Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules that endow new functions to enzymes via proximity-mediated effect are emerging. Phosphorylation (native or neo) of any given protein-of-interest can alter its structure and function, and we hypothesized that such modifications can be accomplished by small molecules that bring a kinase in proximity to the protein-of-interest. Herein, we describe phosphorylation-inducing chimeric small molecules (PHICS), which enable two example kinases-AMPK and PKC-to phosphorylate target proteins that are not otherwise substrates for these kinases.

Structural and functional characterization of oligomeric states of proteins in RecFOR pathway.

RecFOR pathway is the principal repair pathway for double strand break and single strand gap repair in Thermus thermophilus. RecF and RecR exist as monomer and dimer in solution, interestingly; they undergo condition-dependent dimerization and tetramerization, respectively during the DNA break repair. However, their importance in protein-protein and protein-DNA interactions remains elusive.

Profile of the 2016 dengue outbreak in Nepal.

Objective: The objective of this study was to obtain clinical, virological and demographic data detailing the 2016 dengue outbreak in Nepal.

Results: Dengue disease was first reported in Nepal in 2004 and several major outbreaks have occurred since then, with a significant impact on public health. An outbreak of dengue fever occurred in Nepal during June to November 2016, with a peak number of cases reported in September.

Structural and functional roles of dynamically correlated residues in thymidylate kinase.

Thymidylate kinase is an important enzyme in DNA synthesis. It catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, with ATP as the preferred phosphoryl donor, in the presence of Mg. In this study, the dynamics of the active site and the communication paths between the substrates, ATP and TMP, are reported for thymidylate kinase from Thermus thermophilus.

Molecular docking and molecular dynamics simulations of fumarate hydratase and its mutant H235N complexed with pyromellitic acid and citrate.

Fumarase catalyzes the reversible, stereospecific hydration/dehydration of fumarate to L-malate during the Kreb's cycle. In the crystal structure of the tetrameric fumarase, it was found that some of the active site residues S145, T147, N188 G364 and H235 had water-mediated hydrogen bonding interactions with pyromellitic acid and citrate which help to the protonation state for the conversion of fumarate to malate. When His 235 is mutated with Asn (H235N), water-mediated interactions were lost due to the shifting of active site water molecule by 0.

Structural studies of a hyperthermophilic thymidylate kinase enzyme reveal conformational substates along the reaction coordinate.

Unlabelled: Thymidylate kinase (TMK) is a key enzyme which plays an important role in DNA synthesis. It belongs to the family of nucleoside monophosphate kinases, several of which undergo structure-encoded conformational changes to perform their function. However, the absence of three-dimensional structures for all the different reaction intermediates of a single TMK homolog hinders a clear understanding of its functional mechanism.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of thymidylate kinase (TTHA1607) from Thermus thermophilus HB8.

Nucleotide biosynthesis plays a key role in cell survival and cell proliferation. Thymidylate kinase is an enzyme that catalyses the conversion of dTMP to dTDP using ATP-Mg(2+) as a phosphoryl-donor group. This enzyme is present at the junction of the de novo and salvage pathways; thus, any inhibitor designed against it will result in cell death.