Search NASA⌕ Search

Engineering topics

Meagher, Jennifer L.

Publications and source records attributed to Meagher, Jennifer L..

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

A structure-based mechanism for displacement of the HEXIM adapter from 7SK small nuclear RNA

Productive transcriptional elongation of many cellular and viral mRNAs requires transcriptional factors to extract pTEFb from the 7SK snRNP by modulating the association between HEXIM and 7SK snRNA. In HIV-1, Tat binds to 7SK by displacing HEXIM. However, without the structure of the 7SK-HEXIM complex, the constraints that must be overcome for displacement remain unknown. Furthermore, while structure details of the Tat NL4-3 -7SK complex have been elucidated, it is unclear how subtypes with more HEXIM-like Tat sequences accomplish displacement. Here we report the structures of HEXIM, Tat G , and Tat Fin arginine rich motifs in complex with the apical stemloop-1 of 7SK. While most interactions between 7SK with HEXIM and Tat are similar, critical differences exist that guide function. First, the conformational plasticity of 7SK enables the formation of three different base pair configurations at a critical remodeling site, which allows for the modulation required for HEXIM binding and its subsequent displacement by Tat. Furthermore, the specific sequence variations observed in various Tat subtypes all converge on remodeling 7SK at this region. Second, we show that HEXIM primes its own displacement by causing specific local destabilization upon binding — a feature that is then exploited by Tat to bind 7SK more efficiently.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Targeted disruption of pi–pi stacking in Malaysian banana lectin reduces mitogenicity while preserving antiviral activity

Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi–pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi–pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi–pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi–pi stacking without disrupting the overall lectin structure. These findings show that pi–pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structure of the zinc-finger antiviral protein in complex with RNA reveals a mechanism for selective targeting of CG-rich viral sequences

Infection of animal cells by numerous viruses is detected and countered by a variety of means, including recognition of nonself nucleic acids. The zinc finger antiviral protein (ZAP) depletes cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared with endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, 4-zinc finger human (h) ZAP RNA-binding domain (RBD) and a CG dinucleotide-containing RNA target. We report the structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the 4 zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, cross-linking immunoprecipitation sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high-affinity, specific binding to CG dinucleotide-containing RNA. Moreover, variations in RNA-binding specificity among a panel of CG-binding pocket mutants quantitatively predict their selective antiviral activity against a CG-enriched HIV-1 strain. Overall, the hZAP RBD RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

A Potent and Selective Small-Molecule Degrader of STAT3 Achieves Complete Tumor Regression In Vivo

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. Here we report the discovery of SD-36, a small-molecule degrader of STAT3. SD-36 potently induces the degradation of STAT3 protein in vitro and in vivo and demonstrates high selectivity over other STAT members. Induced degradation of STAT3 results in a strong suppression of its transcription network in leukemia and lymphoma cells. SD-36 inhibits the growth of a subset of acute myeloid leukemia and anaplastic large-cell lymphoma cell lines by inducing cell-cycle arrest and/or apoptosis. SD-36 achieves complete and long-lasting tumor regression in multiple xenograft mouse models at well-tolerated dose schedules. Degradation of STAT3 protein, therefore, is a promising cancer therapeutic strategy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Structure-Based Discovery of CF53 as a Potent and Orally Bioavailable Bromodomain and Extra-Terminal (BET) Bromodomain Inhibitor

We report the structure-based discovery of CF53 (28) as a highly potent and orally active inhibitor of bromodomain and extra-terminal (BET) proteins. By the incorporation of a NH-pyrazole group into the 9H-pyrimido[4,5-b]indole core, we identified a series of compounds that bind to BRD4 BD1 protein with K i values of <1 nM and achieve low nanomolar potencies in the cell growth inhibition of leukemia and breast cancer cells. The most-promising compound, CF53, possesses excellent oral pharmacokinetic properties and achieves significant antitumor activity in both triple-negative breast cancer and acute leukemia xenograft models in mice. Determination of the co-crystal structure of CF53 with the BRD4 BD1 protein provides a structural basis for its high binding affinity to BET proteins. CF53 is very selective over non-BET bromodomain-containing proteins. Furthermore, these data establish CF53 as a potent, selective, and orally active BET inhibitor, which warrants further evaluation for advanced preclinical development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Structure-Based Discovery of 4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9 H -pyrimido[4,5- b ]indol-7-yl)-3,5-dimethylisoxazole (CD161) as a Potent and Orally Bioavailable BET Bromodomain Inhibitor

We have designed and synthesized 9H-pyrimido[4,5-b]indole-containing compounds to obtain potent and orally bioavailable BET inhibitors. By incorporation of an indole or a quinoline moiety to the 9H-pyrimido[4,5-b]indole core, we identified a series of small molecules showing high binding affinities to BET proteins and low nanomolar potencies in inhibition of cell growth in acute leukemia cell lines. One such compound, 4-(6-methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (31) has excellent microsomal stability and good oral pharmacokinetics in rats and mice. Orally administered, 31 achieves significant antitumor activity in the MV4;11 leukemia and MDA-MB-231 triple-negative breast cancer xenograft models in mice. Determination of the cocrystal structure of 31 with BRD4 BD2 provides a structural basis for its high binding affinity to BET proteins. Testing its binding affinities against other bromodomain-containing proteins shows that 31 is a highly selective inhibitor of BET proteins. Here, our data show that 31 is a potent, selective, and orally active BET inhibitor.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)

Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively redistribute S-palmitoylated proteins toward different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The depalmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein, we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, the trifluoromethyl group of ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Conformation-Selective Analogues of Dasatinib Reveal Insight into Kinase Inhibitor Binding and Selectivity

In the kinase field, there are many widely held tenets about conformation-selective inhibitors that have yet to be validated using controlled experiments. As such, we have designed, synthesized, and characterized a series of kinase inhibitor analogues of dasatinib, an FDA-approved kinase inhibitor that binds the active conformation. This inhibitor series includes two Type II inhibitors that bind the DFG-out inactive conformation and two inhibitors that bind the αC-helix-out inactive conformation. Using this series of compounds, we analyze the impact that conformation-selective inhibitors have on target binding and kinome-wide selectivity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗