32 research outputs found
Synthesis and evaluation of troponoids as a new class of antibiotics
Novel antibiotics
are urgently needed. The troponoids [tropones,
tropolones, and α-hydroxytropolones (α-HT)] can have anti-bacterial
activity. We synthesized or purchased 92 troponoids and evaluated
their antibacterial activities against Staphylococcus
aureus, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. Preliminary hits were assessed
for minimum inhibitory concentrations (MIC80) and cytotoxicity
(CC50) against human hepatoma cells. Sixteen troponoids
inhibited S. aureus/E. coli/A. baumannii growth by â„80% growth at 50 values >50 ÎŒM. Two selected tropolones (63 and 285) inhibited 18 methicillin-resistant S.
aureus (MRSA) strains with similar MIC80 values as against a reference strain. Two selected thiotropolones
(284 and 363) inhibited multidrug-resistant
(MDR) E. coli with MIC80 â€30 ÎŒM. One α-HT (261) inhibited
MDR-A. baumannii with MIC80 â€30 ÎŒM. This study opens new avenues for development
of novel troponoid antibiotics to address the critical need to combat
MDR bacterial infections
Chemical Control over Immune Recognition: A Class of Antibody-Recruiting Small Molecules That Target Prostate Cancer
[Image: see text] Prostate cancer is the second leading cause of cancer-related death among the American male population, and society is in dire need of new approaches to treat this disease. Here we report the design, synthesis, and biological evaluation of a class of bifunctional small molecules, called antibody-recruiting molecules targeting prostate-cancer (ARM-Ps), that enhance the recognition of prostate cancer cells by the human immune system. ARM-P derivatives were designed rationally via the computational analysis of crystallographic data, and we demonstrate here that these materials are able to: (1) bind PSMA with high affinity (high pM to low nM), (2) template the formation of ternary complexes between anti-DNP antibodies, ARM-P, and LNCaP human prostate cancer cells, and (3) mediate the antibody-dependent killing of LNCaP cells in the presence of human effector cells. This manuscript describes the application of fundamental chemical principles to the design of a novel class of molecules with high therapeutic potential. We believe that this general small-molecule-based strategy could give rise to novel directions in treating cancer and other diseases
Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism
Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 A° . Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator -thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiledmechanistic details of the two-metalion catalysis at atomic resolution
3,7-Dihydroxytropolones Inhibit Initiation of Hepatitis B Virus Minus-Strand DNA Synthesis
Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with epsilon (Δ), a cis-acting regulatory signal located at the 5â terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to Δ is necessary for pgRNA encapsidation and synthesis of a short primer covalently attached to its terminal domain. Although we identified small molecules that recognize HBV Δ RNA, these failed to inhibit protein-primed DNA synthesis. However, since initiation of HBV (-) strand DNA synthesis occurs within a complex of viral and host components (e.g., Hsp90, DDX3 and APOBEC3G), we considered an alternative therapeutic strategy of allosteric inhibition by disrupting the initiation complex or modifying its topology. To this end, we show here that 3,7-dihydroxytropolones (3,7-dHTs) can inhibit HBV protein-primed DNA synthesis. Since DNA polymerase activity of a ribonuclease (RNase H)-deficient HBV reverse transcriptase that otherwise retains DNA polymerase function is also abrogated, this eliminates direct involvement of RNase (ribonuclease) H activity of HBV reverse transcriptase and supports the notion that the HBV initiation complex might be therapeutically targeted. Modeling studies also provide a rationale for preferential activity of 3,7-dHTs over structurally related α-hydroxytropolones (α-HTs)
Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors
The natural product α-hydroxytropolones manicol and ÎČ-thujaplicinol inhibit replication of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively) at nontoxic concentrations. Because these were originally developed as divalent metal-sequestering inhibitors of the ribonuclease H activity of HIV-1 reverse transcriptase, α-hydroxytropolones likely target related HSV proteins of the nucleotidyltransferase (NTase) superfamily, which share an âRNase H-likeâ fold. One potential candidate is pUL15, a component of the viral terminase molecular motor complex, whose C-terminal nuclease domain, pUL15C, has recently been crystallized. Crystallography also provided a working model for DNA occupancy of the nuclease active site, suggesting potential proteinânucleic acid contacts over a region of âŒ14 bp. In this work, we extend crystallographic analysis by examining pUL15C-mediated hydrolysis of short, closely related DNA duplexes. In addition to defining a minimal substrate length, this strategy facilitated construction of a dual-probe fluorescence assay for rapid kinetic analysis of wild-type and mutant nucleases. On the basis of its proposed role in binding the phosphate backbone, studies with pUL15C variant Lys700Ala showed that this mutation affected neither binding of duplex DNA nor binding of small molecule to the active site but caused a 17-fold reduction in the turnover rate (kcat), possibly by slowing conversion of the enzymeâsubstrate complex to the enzymeâproduct complex and/or inhibiting dissociation from the hydrolysis product. Finally, with a view of pUL15-associated nuclease activity as an antiviral target, the dual-probe fluorescence assay, in combination with differential scanning fluorimetry, was used to demonstrate inhibition by several classes of small molecules that target divalent metal at the active site
A Remote Arene-Binding Site on Prostate Specific Membrane Antigen Revealed by Antibody-Recruiting Small Molecules
Prostate specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase overexpressed in many forms of prostate cancer. Our laboratory has recently disclosed a class of small molecules, called ARM-Ps (antibody-recruiting molecule targeting prostate cancer) that are capable of enhancing antibody-mediated immune recognition of prostate cancer cells. Interestingly, during the course of these studies, we found ARM-Ps to exhibit extraordinarily high potencies toward PSMA, compared to previously reported inhibitors. Here, we report in-depth biochemical, crystallographic, and computational investigations which elucidate the origin of the observed affinity enhancement. These studies reveal a previously unreported arene-binding site on PSMA, which we believe participates in an aromatic stacking interaction with ARMs. Although this site is composed of only a few amino acid residues, it drastically enhances small molecule binding affinity. These results provide critical insights into the design of PSMA-targeted small molecules for prostate cancer diagnosis and treatment; more broadly, the presence of similar arene-binding sites throughout the proteome could prove widely enabling in the optimization of small-moleculeâprotein interactions
An Oxidopyrylium Cyclization/Ring-Opening Route to Polysubstituted 뱉Hydroxytropolones
α-Hydroxytropolones are a class of molecules with therapeutic potential against several human diseases. However, structureâactivity relationship studies on these molecules have been limited due to a scarcity of efficient synthetic methods to access them. It is demonstrated herein that α-hydroxytropolones can be generated through a BCl<sub>3</sub>-mediated ring-opening/aromatization/demethylation process on 8-oxabicyclo[3.2.1]octenes. Used in conjunction with an improved method based on established oxidopyrylium dipolar cycloadditions, several polysubstituted α-hydroxytropolones can be accessed in three steps from readily available α-hydroxy-Îł-pyrones
Free Energy-Based Virtual Screening and Optimization of RNase H Inhibitors of HIVâ1 Reverse Transcriptase
We
report the results of a binding free energy-based virtual screening
campaign of a library of 77 α-hydroxytropolone derivatives against
the challenging RNase H active site of the reverse transcriptase (RT)
enzyme of human immunodeficiency virus-1. Multiple protonation states,
rotamer states, and binding modalities of each compound were individually
evaluated. The work involved more than 300 individual absolute alchemical
binding free energy parallel molecular dynamics calculations and over
1 million CPU hours on national computing clusters and a local campus
computational grid. The thermodynamic and structural measures obtained
in this work rationalize a series of characteristics of this system
useful for guiding future synthetic and biochemical efforts. The free
energy model identified key ligand-dependent entropic and conformational
reorganization processes difficult to capture using standard docking
and scoring approaches. Binding free energy-based optimization of
the lead compounds emerging from the virtual screen has yielded four
compounds with very favorable binding properties, which will be the
subject of further experimental investigations. This work is one of
the few reported applications of advanced-binding free energy models
to large-scale virtual screening and optimization projects. It further
demonstrates that, with suitable algorithms and automation, advanced-binding
free energy models can have a useful role in early-stage drug-discovery
programs
Catalytic Enantioselective Intermolecular [5 + 2] Dipolar Cycloadditions of a 3âHydroxy-4-pyrone-Derived Oxidopyrylium Ylide
The
first catalytic enantioselective [5 + 2] dipolar cycloaddition
of a 3-hydroxy-4-pyrone-derived oxidopyrylium ylide is described.
These studies leveraged the recently recognized ability of oxidopyrylium
dimers to serve as the source of ylide, which was found to be key
to increasing yields and achieving enantiomeric excesses up to 99%.
General reaction conditions were identified for an array of α,ÎČ-unsaturated
aldehyde dipolarophiles. Reaction products possess four stereocenters,
and subsequent reduction introduced a fifth contiguous stereocenter
with total stereocontrol