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 (ε), a cis-acting regulatory signal located at the 5’ terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to ε 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 ε 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 β-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 “RNase H-like” 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 protein–nucleic acid contacts over a region of ∼14 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 enzyme–substrate complex to the enzyme–product 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-molecule–protein 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 “on-the-fly” 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

Effect of biliopancreatic diversion on hypertension in severely obese patients.

Hypertension is a medical disorder frequently associated with severe obesity, and the effect of weight loss on the reduction of blood pressure has been well established. In this study, the relationships between the weight loss surgically obtained by biliopancreatic diversion and blood pressure were investigated in a population of severely obese patients with preoperative hypertension. At 1 year following the operation, blood pressure was normalized in more than half of patients; in a further 10% of cases the hypertensive status resolved within the 3-year follow-up period. The resolution of hypertension was independently associated with age and body weight and was unrelated to sex, the amount of weight loss, or body fat distribution. In severely obese patients with hypertension undergoing bariatric surgery, biliopancreatic diversion is advisable since it achieves and supports the maintenance of body weight close to the ideal value

A 15-year evaluation of biliopancreatic diversion according to the Bariatric Analysis Reporting Outcome System (BAROS)

BACKGROUND: Biliopancreatic diversion (BPD) is the most effective bariatric procedure in terms of weight loss. However, analysis of the quality of life (QoL) has never been reported. The BAROS, giving a score to each operated patient on weight loss, improvement in medical conditions, QoL, complications and reoperations, has proven to be a standard reference for evaluating bariatric surgery outcomes. METHODS: In order to apply the BAROS to BPD, we sent a questionnaire to 1,800 BPD patients who had been operated between 1984 and 1998. The response-rate was 51.2%. Out of 1,709 questionnaires which actually reached their destination, we had 858 fully compiled returned. There were 615 women. 596 patients had had an ad hoc stomach (AHS) BPD, and 262 had had an ad hoc stomach ad hoc alimentary limb (AHS-AHAL) BPD. RESULTS: According to the scoring key, 3.5% were classified as a failure, 11% were fair results, 22.8% good, 39.5% very good, and 23.2% excellent results. Considering AHS BPD and AHS-AHAL BPD separately,while the mean excess weight percent loss was 70.5+/-23 and 64.7+/-17 respectively, the failure rate was 6% in the first group and 2% in the AHAL group, while 11% and 6% of cases respectively were fair results, 24% and 20% good, 36% and 47% very good, 23% and 25% excellent results. CONCLUSION: The BAROS evaluation of BPD highlights the importance of its flexibility: the new policy of adapting the procedure to individual characteristics caused a drop in the failure rate and an increase in good, very good and excellent results