73 research outputs found
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
A Collective Variable for the Rapid Exploration of Protein Druggability
An efficient molecular simulation
methodology has been developed
for the evaluation of the druggability (ligandability) of a protein.
Previously proposed techniques were designed to assess the druggability
of crystallographic structures and cannot be tightly coupled to molecular
dynamics (MD) simulations. By contrast, the present approach, JEDI
(<u>J</u>ust <u>E</u>xploring <u>D</u>ruggability at protein <u>I</u>nterfaces),
features a druggability potential made of a combination of empirical
descriptors that can be collected âon-the-flyâ during
MD simulations. Extensive validation studies indicate that JEDI analyses
discriminate druggable and nondruggable protein binding site conformations
with accuracy similar to alternative methodologies, and at a fraction
of the computational cost. Since the JEDI function is continuous and
differentiable, the druggability potential can be used as collective
variable to rapidly detect cryptic druggable binding sites in proteins
with a variety of MD free energy methods. Protocols for applications
to flexible docking problems are outlined
Il rapporto tra intertestualit\ue0 e intercultura nell\u2019insegnamento dell\u2019italiano come lingua straniera: una proposta
L\u2019applicazione del portfolio elettronico EPOS in corsi d\u2019italiano per studenti non specialisti: primi risultati
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