SummaryRhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

A fundamental motif in canonical nucleic acid structure is the base pair. Mutations that disrupt base pairs are typically destabilizing, but stability can often be restored by a second mutation that replaces the original base pair with an isosteric variant. Such concerted changes are a way to identify helical regions in secondary structures and to identify new functional motifs in sequenced genomes. In principle, such analysis can be extended to non-canonical nucleic acid structures, but this approach has not been utilized because the sequence requirements of such structures are not well understood. Here we investigate the sequence requirements of a G-quadruplex that can both bind GTP and promote peroxidase reactions. Characterization of all 256 variants of the central tetrad in this structure indicates that certain mutations can compensate for canonical G-G-G-G tetrads in the context of both GTP-binding and peroxidase activity. Furthermore, the sequence requirements of these two motifs are significantly different, indicating that tetrad sequence plays a role in determining the biochemical specificity of G-quadruplex activity. Our results provide insight into the sequence requirements of G-quadruplexes, and should facilitate the analysis of such motifs in sequenced genomes.

Functional DNAm olecules are useful components in nanotechnology and synthetic biology.T oexpand the toolkit of functional DNAp arts,i nt his study we used artificial evolution to identify ag lowing deoxyribozyme called Supernova. This deoxyribozyme transfers ap hosphate from a1 ,2dioxetane substrate to its 5' hydroxylg roup,w hich triggers ac hemiluminescent reaction and af lash of blue light. An engineered version of Supernova is only catalytically active in the presence of an oligonucleotide complementary to its 3' end, demonstrating that light production can be coupled to ligand binding.W eanticipate that Supernova will be useful in awide variety of applications,i ncluding as as ignaling component in allosterically regulated sensors and in logic gates of molecular computers.

Functional DNAm olecules are useful components in nanotechnology and synthetic biology.T oexpand the toolkit of functional DNAp arts,i nt his study we used artificial evolution to identify ag lowing deoxyribozyme called Supernova. This deoxyribozyme transfers ap hosphate from a1 ,2dioxetane substrate to its 5' hydroxylg roup,w hich triggers ac hemiluminescent reaction and af lash of blue light. An engineered version of Supernova is only catalytically active in the presence of an oligonucleotide complementary to its 3' end, demonstrating that light production can be coupled to ligand binding.W eanticipate that Supernova will be useful in awide variety of applications,i ncluding as as ignaling component in allosterically regulated sensors and in logic gates of molecular computers.

Supernova is a chemiluminescent deoxyribozyme recently discovered in our group. It transfers the phosphate group from the 1,2-dioxetane substrate CDP-Star to its 5' hydroxyl group, which triggers a decomposition reaction and the production of light. Here we investigated the effects of reaction conditions on the ability of Supernova to generate a chemiluminescent signal (using a plate reader assay) and to phosphorylate itself (using a ligation assay). Our experiments indicate that multiple zinc ions are required for catalytic function, suggesting links between Supernova and protein enzymes that catalyze similar reactions. They also show how factors such as pH, potassium concentration, CDP-Star concentration, and DNA concentration affect the reaction. By combining information from different experiments, the rate enhancement of light production was increased by more than 1000-fold. These results should be useful for applications in which Supernova is used as a sensor.

G-quadruplexes are noncanonical nucleic acid structures formed by stacked guanine tetrads. They are capable of a range of functions and thought to play widespread biological roles. This diversity raises an important question: what determines the biochemical specificity of G-quadruplex structures? The answer is particularly important from the perspective of biological regulation because genomes can contain hundreds of thousands of G-quadruplexes with a range of functions. Here we analyze the specificity of each sequence in a 496-member library of variants of a reference G-quadruplex with respect to five functions. Our analysis shows that the sequence requirements of G-quadruplexes with these functions are different from one another, with some mutations altering biochemical specificity by orders of magnitude. Mutations in tetrads have larger effects than mutations in loops, and changes in specificity are correlated with changes in multimeric state. To complement our biochemical data we determined the solution structure of a monomeric G-quadruplex from the library. The stacked and accessible tetrads rationalize why monomers tend to promote a model peroxidase reaction and generate fluorescence. Our experiments support a model in which the sequence requirements of G-quadruplexes with different functions are overlapping but distinct. This has implications for biological regulation, bioinformatics, and drug design.

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