scholarly journals Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

2020 ◽  
Author(s):  
Hanieh Ghassabian ◽  
Federico Falchi ◽  
Veronica Di Antonio ◽  
Martina Timmoneri ◽  
Beatrice Mercorelli ◽  
...  
Keyword(s):  
Small Molecules ◽  
Dna Polymerase ◽  
Therapeutic Intervention ◽  
Catalytic Subunit ◽  
Viral Gene ◽  
Drug Resistant ◽  
New Targets ◽  
New Class ◽  
Processivity Factor

ABSTRACTHuman cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite the availability of several antiviral drugs, their utility is limited by poor bioavailability, toxicity, and resistant strains emergence. Therefore, it is crucial to identify new targets of therapeutic intervention. The dimerization of HCMV DNA polymerase processivity factor UL44 plays an essential role in the viral life cycle being required for oriLyt-dependent DNA replication. We validated the existence of UL44 homodimers both in vitro and in living cells by a variety of approaches, including GST pulldown, thermal shift, FRET and BRET assays. Dimerization occurred with an affinity comparable to that of the UL54/UL44 interaction, and was impaired by amino acid substitutions at the dimerization interface. Subsequently, we performed an in-silico screening to select 18 small molecules (SMs) potentially interfering with UL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the 18 selected SMs led to the identification of four active SMs. The most active one also inhibited AD169 in plaque reduction assays, and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, treatment of infected cells specifically reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA synthesis. Therefore, SMs inhibitors of UL44 dimerization could represent a new class of HCMV inhibitors, alternative to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.IMPORTANCEHCMV is a ubiquitous infectious agent causing life-lasting infections in humans. HCMV primary infections and reactivation in non-immunocompetent individuals often result in life-threatening conditions. Antiviral therapy mainly targets the DNA polymerase catalytic subunit UL54 and is often limited by toxicity and selection of drug-resistant viral strains, making the identification of new targets of therapeutic intervention crucial for a successful management of HCMV infections. The significance of our work is in identifying the dimerization of the DNA polymerase processivity factor UL44 as an alternative antiviral target. We could show that full length UL44 dimerizes in a cellular context with high affinity and that such interaction could be targeted by small molecules, thus inhibiting the replication of several HCMV strains, including a drug-resistant mutant. Thus, our work could pave the way to the development of a new class of anti-HCMV compounds that act by targeting UL44 dimerization.

scholarly journals Divide et Impera: An In Silico Screening Targeting HCMV ppUL44 Processivity Factor Homodimerization Identifies Small Molecules Inhibiting Viral Replication

2020 ◽  
Author(s):  
Hanieh Ghassabian ◽  
Federico Falchi ◽  
Martina Timmoneri ◽  
Beatrice Mercorelli ◽  
Arianna Loregian ◽  
...  
Keyword(s):  
Small Molecules ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
In Silico ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Protein Protein Interactions ◽  
In Silico Screening ◽  
Infected Cells ◽  
Processivity Factor

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets of therapeutic intervention. Among the latter, viral protein-protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle being required for oriLyt-dependent DNA replication, we performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB40-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited AD169 in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, treatment of infected cells with B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA synthesis. Therefore, inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.

scholarly journals Divide et impera: An In Silico Screening Targeting HCMV ppUL44 Processivity Factor Homodimerization Identifies Small Molecules Inhibiting Viral Replication

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 941
Author(s):  
Hanieh Ghassabian ◽  
Federico Falchi ◽  
Martina Timmoneri ◽  
Beatrice Mercorelli ◽  
Arianna Loregian ◽  
...  
Keyword(s):  
Small Molecules ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Post Infection ◽  
In Silico ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Protein Protein Interactions ◽  
In Silico Screening ◽  
Processivity Factor

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS patients and transplant recipients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets for therapeutic intervention. Among the latter, viral protein–protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle, being required for oriLyt-dependent DNA replication, it can be considered a potential therapeutic target. We therefore performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited HCMV AD169 strain in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with the inhibition of viral DNA synthesis measured by qPCR starting from 72 h post infection. Therefore, our data suggest that inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

scholarly journals Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Nadine Lemaître ◽  
Xiaofei Liang ◽  
Javaria Najeeb ◽  
Chul-Jin Lee ◽  
Marie Titecat ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Biosynthetic Pathway ◽  
Lipid A ◽  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
Drug Resistant ◽  
Bubonic Plague ◽  
Gram Negative ◽  
New Class

ABSTRACT The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis. Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria. IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains. IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

scholarly journals Discovery of a cofactor-independent inhibitor ofMycobacterium tuberculosisInhA

2018 ◽  
Vol 1 (3) ◽  
pp. e201800025 ◽  
Author(s):  
Yi Xia ◽  
Yasheen Zhou ◽  
David S Carter ◽  
Matthew B McNeil ◽  
Wai Choi ◽  
...  
Keyword(s):  
Oral Delivery ◽  
Drug Exposure ◽  
Drug Resistant ◽  
Antitubercular Agents ◽  
Independent Manner ◽  
Structural Investigations ◽  
New Class ◽  
Enoyl Acp Reductase ◽  
Drug Resistant Strains

New antitubercular agents are needed to combat the spread of multidrug- and extensively drug-resistant strains ofMycobacterium tuberculosis. The frontline antitubercular drug isoniazid (INH) targets the mycobacterial enoyl-ACP reductase, InhA. Resistance to INH is predominantly through mutations affecting the prodrug-activating enzyme KatG. Here, we report the identification of the diazaborines as a new class of direct InhA inhibitors. The lead compound, AN12855, exhibited in vitro bactericidal activity against replicating bacteria and was active against several drug-resistant clinical isolates. Biophysical and structural investigations revealed that AN12855 binds to and inhibits the substrate-binding site of InhA in a cofactor-independent manner. AN12855 showed good drug exposure after i.v. and oral delivery, with 53% oral bioavailability. Delivered orally, AN12855 exhibited dose-dependent efficacy in both an acute and chronic murine model of tuberculosis infection that was comparable with INH. Combined, AN12855 is a promising candidate for the development of new antitubercular agents.

scholarly journals The celecoxib derivative AR-12 has broad spectrum antifungal activity in vitro and improves the activity of fluconazole in a murine model of cryptococcosis

2016 ◽  
pp. AAC.01061-16 ◽  
Author(s):  
Kristy Koselny ◽  
Julianne Green ◽  
Louis DiDone ◽  
Justin P. Halterman ◽  
Annette W. Fothergill ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Small Molecules ◽  
Broad Spectrum ◽  
Murine Model ◽  
Antifungal Drugs ◽  
Dimorphic Fungi ◽  
New Class ◽  
Cancer Agent

Only one new class of antifungal drugs has been introduced into clinical practice in the last thirty years and, thus, the identification of small molecules with novel mechanisms of action is an important goal of current anti-infective research. Here, we describe the characterization of the spectrum of in vitro activity and in vivo activity of AR-12, a celecoxib-derivative which has been tested in a Phase I clinical trial as an anti-cancer agent. AR-12 inhibits fungal acetyl CoA synthetase in vitro and is fungicidal at concentrations similar to those achieved in human plasma. AR-12 has a broad spectrum of activity including active against yeasts (e.g.,C. albicans, non-albicansCandidaspp.,C. neoformans); molds (e.g.,Fusarium,Mucor), and dimorphic fungi (Blastomyces,Histoplasma, andCoccidioides) with minimum inhibitory concentrations of 2-4 μg/mL. AR-12 is also active against azole- and echinocandin-resistantCandidaisolates and sub-inhibitory AR-12 concentrations increase susceptibility of fluconazole- and echinocandin-resistantCandidaisolates. Finally, AR-12 also increases the activity of fluconazole in a murine model of cryptococcosis. Taken together, these data indicate that AR-12 represents a promising class of small molecules with broad spectrum antifungal activity.

Synthesis and antimalarial activity of new 4-aminoquinolines active against drug resistant strains

RSC Advances ◽  
2016 ◽  
Vol 6 (107) ◽  
pp. 105676-105689 ◽  
Author(s):  
Srinivasarao Kondaparla ◽  
Awakash Soni ◽  
Ashan Manhas ◽  
Kumkum Srivastava ◽  
Sunil K. Puri ◽  
...  
Keyword(s):  
Antimalarial Activity ◽  
Drug Resistant ◽  
New Class ◽  
Resistant Strains ◽  
Drug Resistant Strains

In the present study we have synthesized a new class of 4-aminoquinoline derivatives and bioevaluated them for antimalarial activity against theP. falciparum in vitro(3D7 & K1) andP. yoelii in vivo(N-67 strain).

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