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Dysregulation of master regulator c - MYC (MYC) plays a central role in hepatocellular carcinoma (HCC) and other cancers but remains an elusive target for therapeutic intervention. MYC expression is epigenetically modulated within naturally occurring DNA loop structures, Insulated Genomic Domains (IGDs). We present a therapeutic approach using an epigenomic controller (EC), a programmable epigenomic mRNA medicine, to precisely modify MYC IGD sub-elements, leading to methylation of MYC regulatory elements and durable downregulation of MYC mRNA transcription. Significant antitumor activity is observed in preclinical models of HCC treated with the MYC-targeted EC, as monotherapy or in combination with tyrosine kinase or immune checkpoint inhibitors. These findings pave the way for clinical development of MYC-targeting epigenomic controllers in HCC patients and provide a framework for programmable epigenomic mRNA therapeutics for cancer and other diseases. Development of targeted MYC inhibitors for cancer therapy remains challenging. Here, the authors design an mRNA medicine which downregulates MYC gene transcription via epigenetic modification of MYC regulatory elements, showing significant antitumor activity in preclinical models of hepatocellular carcinoma.

Pathogenic gene dysregulation can be attributed to chromatin state change that pre-transcriptionally regulates expression. Recent breakthroughs elucidating the rules governing this DNA control layer, an epigenetic code, unlock a modality in precision medicine to target gene dysregulation across myriad diseases. Here we present a modular platform to design programmable mRNA therapeutics, Epigenomic Controllers (EC), that control gene expression through directed epigenetic change. By leveraging natural mechanisms, ECs tune expression levels of one or multiple genes with durable effect of weeks-to-months in female mice following a single dose. We design and characterize ECs to multiple target genes and identify an EC that effectively inhibits the cancer- and inflammatory-disorder-associated multi-gene cluster CXCL1-8 . With precision targeting of NF-kB signaling and identification of homologous murine surrogates, ECs significantly reduce neutrophil migration in vivo during acute lung inflammation in female mice. A platform approach to EC design for epigenomic modulation expands treatment frontiers for diverse gene targets, including those considered “undruggable.” mRNA medicines hold enormous promise for drugging targets outside the reach of classic small molecules. Here, the authors develop a modular platform to design programmable mRNA therapeutics that act by modifying epigenetic state.

Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

Significance Here, we examine the activity profile of the haloalkanoic acid dehalogenase (HAD) superfamily by screening a customized library against >200 enzymes from a broad sampling of the superfamily. From this dataset, we can infer the function of nearly 35% of the superfamily. Overall, the superfamily was found to show high substrate ambiguity, with 75% of the superfamily utilizing greater than five substrates. In addition, the HAD members with the least amount of structural accessorization of the Rossmann fold were found to be the most specific, suggesting that elaboration of the core domain may have led to increased substrate range of the superfamily. Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.

Messenger RNA (mRNA) is a promising new class of therapeutics that has potential for treatment of diseases in fields such as immunology, oncology, vaccines, and inborn errors of metabolism. mRNA therapy has several advantages over DNA-based gene therapy, including the lack of the need for nuclear import and transcription, as well as limited possibility of genomic integration. One drawback of mRNA therapy, especially in cases such as metabolic disorders where repeated dosing will be necessary, is the relatively short in vivo half-life of mRNA (∼6–12 h). We hypothesize that protein engineering designed to improve translation, yielding longer-lasting protein, or modifications that would increase enzymatic activity would be helpful in alleviating this issue. In this study, we present two examples where sequence engineering improved the expression and duration, as well as enzymatic activity of target proteins in vitro . We then confirmed these findings in wild-type mice. This work shows that rational engineering of proteins can lead to improved therapies in vivo .

Early developmental treatment of rats with 3,4-methylenedioxymethamphetamine (MDMA) was previously found to cause an abnormal pattern of forebrain serotonergic axon density in adulthood consisting of a cortical hypoinnervation and a striatal hyperinnervation. The present study tested the hypothesis that this reorganization was due to regional differences in brain-derived neurotrophic factor (BDNF) expression. Rats received MDMA (10 mg/kg, s.c., b.i.d.) on postnatal days (PD) 1–4, after which brain tissues were collected on PD 11, 30, and 67 for analysis. BDNF protein levels were found to be elevated in the occipital cortex but not in the hippocampus or striatum following MDMA administration. Serotonin transporter binding (an index of serotonergic fiber integrity) was significantly reduced in the hippocampus at PD 11 but returned to normal by PD 30, whereas the cortex exhibited a delayed reduction that was not manifested until PD 30. These results do not support the view that a region-specific enhancement in BDNF expression mediates the abnormal serotonergic reinnervation observed following neonatal MDMA exposure.

Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

Legionella pneumophila (Lp) is a gram-negative pathogen that is the causative agent of Legionnaire's disease. Once endocytosed by alveolar macrophages, Lp subverts the endosome-lysosome pathway and converts the endosome into an ER-like compartment where the bacterium replicates. Conversion of the endosome into an ER-like compartment requires ∼26 Dot/Icm genes that encode a Type IVb secretion system (T4bSS). The T4bSS is evolutionarily-related to the Incl plasmid conjugation system and also has 7 novel genes. IcmR and icmQ are novel genes and mutation studies revealed that they are essential for intracellular replication. IcmQ is composed of two domains: an N-terminal domain (Qn) and a C-terminal domain (Qc). The Qn domain interacts with the middle region of IcmR (Rm). A previous structure of Rm-Qn interacting domains revealed a four helix bundle (4HB), in which two helices each are contributed respectively by IcmR and IcmQ. In this thesis, crystals of the Rm-IcmQ complex were obtained and the structure was solved by single-wavelength anomalous dispersion (SAD) phasing with selenomethionine labeled complexes. Remarkably, the Qc domain in the IcmR-IcmQ complex has a fold similar to NAD-binding domains in bacterial toxins, such as cholera, diphtheria and pertussis toxins. Each of these toxins exhibits ADP-ribosyltransferase activity; hence, the structure of Qc suggests that it may also have this activity. In support of this idea, binding studies indicated that the Qc domain interacts with NAD. In addition, highly conserved residues in the Qc domain that may play a role in NAD binding were identified by comparative modeling. This included residues that may interact with nicotinamide, adenine and phosphate groups of NAD. Importantly, aspartate 151 is predicted to play a critical role in destabilizing the Nglycosidic bond between the nicotinamide and ribose rings. Further studies are now needed to characterize NAD binding and to identify the substrate of the IcmR-IcmQ complex. This substrate is predicted to function in the T4bSS and ADP-ribosylation by the Qc domain may promote assembly of the T4bSS or regulate its function. This work points the way to obtain a deeper understanding of IcmR-IcmQ function in the T4bSS and suggests a drug target for treating Legionella infections.