Serendipitous Identification of Azine Anticancer Agents Using a Privileged Scaffold Morphing Strategy.

The use of privileged scaffolds as a starting point for the construction of libraries of bioactive compounds is a widely used strategy in drug discovery and development. Scaffold decoration, morphing and hopping are additional techniques that enable the modification of the chosen privileged framework and better explore the chemical space around it. In this study, two series of highly functionalized pyrimidine and pyridine derivatives were synthesized using a scaffold morphing approach consisting of triazine compounds obtained previously as antiviral agents. Newly synthesized azines were evaluated against lymphoma, hepatocarcinoma, and colon epithelial carcinoma cells, showing in five cases acceptable to good anticancer activity associated with low cytotoxicity on healthy fibroblasts. Finally, ADME in vitro studies were conducted on the best derivatives of the two series showing good passive permeability and resistance to metabolic degradation.

In Vitro Combinatorial Activity of Direct Acting Antivirals and Monoclonal Antibodies against the Ancestral B.1 and BQ.1.1 SARS-CoV-2 Viral Variants.

Combination antiviral therapy may be helpful in the treatment of SARS-CoV-2 infection; however, no clinical trial data are available, and combined use of direct-acting antivirals (DAA) and monoclonal antibodies (mAb) has been reported only anecdotally. To assess the cooperative effects of dual drug combinations in vitro, we used a VERO E6 cell-based in vitro system with the ancestral B.1 or the highly divergent BQ.1.1 virus to test pairwise combinations of the licensed DAA, including nirmatrelvir (NRM), remdesivir (RDV) and the active metabolite of molnupiravir (EIDD-1931) as well the combination of RDV with four licensed mAbs (sotrovimab, bebtelovimab, cilgavimab, tixagevimab; tested only with the susceptible B.1 virus). According to SynergyFinder 3.0 summary and weighted scores, all the combinations had an additive effect. Within DAA/DAA combinations, paired scores with the B.1 and BQ.1.1 variants were comparable. In the post hoc analysis weighting synergy by concentrations, several cases of highly synergistic scores were detected at specific drug concentrations, both for DAA/DAA and for RDV/mAb combinations. This was supported by in vitro confirmation experiments showing a more than a linear shift of a drug-effective concentration (IC50) at increasing concentrations of the companion drug, although the effect was prominent with DAA/DAA combinations and minimal or null with RDV/mAb combinations. These results support the cooperative effects of dual drug combinations in vitro, which should be further investigated in animal models before introduction into the clinic.

Switching the three-component Biginelli-like reaction conditions for the regioselective synthesis of new 2-amino[1,2,4]triazolo[1,5-a]pyrimidines.

Among the eight different triazolopyrimidine isomers existing in nature, 1,2,4-triazolo[1,5-a]pyrimidine (TZP) is one of the most studied and used isomers in medicinal chemistry. For some years, our group has been involved in developing regioselective one-pot procedures for the synthesis of 2-amino-7-aryl-5-methyl- and 2-amino-5-aryl-7-methyl-TZPs of interest in the preparation of antiviral agents. In this work, taking advantage of a Biginelli-like multicomponent reaction (MCR), we report the identification of finely tunable conditions to regioselectively synthesize C-6 ester-substituted amino-TZP analogues, both in dihydro and oxidized forms. Indeed, the use of mild acidic conditions is strongly directed toward the regioselective synthesis of 5-aryl-7-methyl C-6-substituted TZP analogues, while the use of neutral ionic liquids shifted the regioselectivity towards 7-aryl-5-methyl derivatives. In addition, the novel synthesized scaffolds were functionalized at the C-2 position and evaluated for their antiviral activity against RNA viruses (influenza virus, flaviviruses, and SARS-CoV-2). Compounds 25 and 26 emerged as promising anti-flavivirus agents, showing activity in the low micromolar range.

Prevalence and phenotypic susceptibility to doravirine of the HIV-1 reverse transcriptase V106I polymorphism in B and non-B subtypes.

AIM: To evaluate the prevalence and in vitro susceptibility to doravirine of RT-V106I polymorphism detected in samples collected from drug-naïve subjects. METHODS: Doravirine susceptibility was measured in site-directed mutants (SDMs) containing V106I, V106A, V106 M and Y188L mutations in subtype B (NL4-3, HXB2) and CRF02_AG background and in recombinant viruses with RT harboring V106I alone derived from 50 PLWH. RESULTS: HIV-1 B subtype was detected in 1523/2705 cases. Prevalence of V106I was 3.2% in B and 2.5% in non-B subtypes, and was higher in subtype F (8.1%), and D (14.3%). Fold-changes (FC) in susceptibility for SDMs were below doravirine biological cutoff (3.0) for V106I, but not for V106A, V106 M, and Y188L. Clinically-derived viruses tested included 22 B (median FC 1.2 [IQR 0.9-1.6]) and 28 non-B subtypes (median FC 1.8 [IQR 0.9-3.0]). Nine (18%) viruses showed FC values equal or higher than the doravirine biological FC cutoff. CONCLUSIONS: The prevalence of the HIV-1 RT-V106I polymorphism in MeditRes HIV consortium remains low, but significantly more prevalent in subtypes D and F. V106I minimally decreased the susceptibility to doravirine in SDMs and most clinical isolates. Reduced susceptibility seems to occur at increased frequency in subtype F1, however the clinical impact remains to be investigated.

HIV-1 Integrase Inhibition Activity by Spiroketals Derived from Plagius flosculosus, an Endemic Plant of Sardinia (Italy) and Corsica (France).

In this work we investigated, for the first time, the effect of Plagius flosculosus (L.) Alavi & Heywood, a Sardinian-Corsican endemic plant, on HIV-1 integrase (IN) activity. The phytochemical analysis of the leaves chloroform extract led us to isolate and characterize three compounds (SPK1, SPK2, and SPK3) belonging to the spiroketals, a group of naturally occurring metabolites of phytochemical relevance with interesting biological properties. Due to their structural diversity, these cyclic ketals have attracted the interest of chemists and biologists. SPK1, SPK2, and SPK3 were evaluated here for their ability to inhibit HIV-1 integrase activity in biochemical assays. The results showed that all the compounds inhibited HIV-1 IN activity. In particular, the most active one was SPK3, which interfered in a low molecular range (IC50 of 1.46 ± 0.16 µM) with HIV-1 IN activity in the presence/absence of the LEDGF cellular cofactor. To investigate the mechanism of action, the three spiroketals were also tested on HIV-1 RT-associated Ribonuclease H (RNase H) activity, proving to be active in inhibiting this function. Although SPK3 was unable to inhibit viral replication in cell culture, it promoted the IN multimerization. We hypothesize that SPK3 inhibited HIV-1 IN through an allosteric mechanism of action.

Nucleoside Derivatives of 2,6-Diaminopurine Antivirals: Base-Modified Nucleosides with Broad-Spectrum Antimicrobial Properties.

The plethora of viral outbreaks experienced in the last decade, together with the widespread distribution of many re-emerging and newly emerging viruses, emphasize the urgent need for novel broad-spectrum antivirals as tools for early intervention in case of future epidemics. Non-natural nucleosides have been at the forefront for the treatment of infectious diseases for many years and still represent one of the most successful classes of antiviral molecules on the market. In the attempt to explore the biologically relevant chemical space of this class of antimicrobials, we describe herein the development of novel base-modified nucleosides by converting previously identified 2,6-diaminopurine antivirals into the corresponding D/L ribonucleosides, acyclic nucleosides and prodrug derivatives. A phenotypic screening against viruses belonging to different families (Flaviviridae, Coronaviridae, Retroviridae) and against a panel of Gram-positive and Gram-negative bacteria, allowed to identify a few interesting molecules with broad-spectrum antimicrobial activities.

Residual phenotypic susceptibility to doravirine in multidrug-resistant HIV-1 from subjects enrolled in the PRESTIGIO Registry.

OBJECTIVES: Doravirine shows a rather distinct resistance profile within the nonnucleoside reverse transcriptase inhibitor (NNRTI) class. This study aimed to evaluate the phenotypic susceptibility to doravirine, rilpivirine and etravirine in a panel of multidrug-resistant (MDR) HIV-1 isolates collected from people living with HIV (PLWH) enrolled in the PRESTIGIO Registry. METHODS: Recombinant viruses expressing PLWH-derived protease, reverse transcriptase coding regions were generated from plasma samples at virological failure with documented resistance to protease inhibitors, nucleoside reverse transcriptase inhibitors, NNRTIs and integrase strand transfer inhibitors. In vitro susceptibility was assessed through a phenotypic assay measuring fold-change values with respect to the reference NL4-3 virus. Genotypic susceptibility was computed by the Stanford HIVdb algorithm 8.9-1. RESULTS: Plasma samples were collected from 22 PLWH: 20 (91%) were male, median age 55 years (IQR 50-58), time since HIV-1 diagnosis 27 years (23-31) and time on antiretroviral treatment 23 years (22-26). Median doravirine, etravirine and rilpivirine fold-change values were 9.8 (2.9-40.4), 42.9 (3.1-100.0) and 100.0 (17.9-100.0), respectively. According to the fold-change cut-offs, full susceptibility was observed in five (23%), four (18%) and one (5%) cases with doravirine, etravirine and rilpivirine, respectively. Irrespective of the presence of specific doravirine mutations, higher numbers of NNRTI mutations correlated with higher fold-change values for doravirine. By comparing the distribution of fold-change values with the Stanford HIVdb predicted susceptibility, a significant correlation was detected for doravirine and rilpivirine but not etravirine. CONCLUSION: Despite extensive cross-resistance among NNRTIs, doravirine can be a valid option in a proportion of PLWH with MDR HIV-1. Doravirine activity appeared to be inferred with fair accuracy by the HIVdb algorithm.

Privileged Scaffold Decoration for the Identification of the First Trisubstituted Triazine with Anti-SARS-CoV-2 Activity.

Current therapy against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are based on the use of Remdesivir 1, Molnupiravir 2, and the recently identified Nirmatrelvir 3. Unfortunately, these three drugs showed some limitations regarding potency and possible drug-drug interactions. A series of derivatives coming from a decoration approach of the privileged scaffold s-triazines were synthesized and evaluated against SAR-CoV-2. One derivative emerged as the hit of the series for its micromolar antiviral activity and low cytotoxicity. Mode of action and pharmacokinetic in vitro preliminary studies further confirm the role as candidates for a future optimization campaign of the most active derivative identified with this work.

Dynamics of Total and Intact HIV-1 DNA in Virologically Suppressed Patients Switching to DTG-Based or ATV-Based Dual Therapy.

BACKGROUND: Clinical trials have demonstrated noninferior viral suppression rates of selected 2-drug regimens (2DRs) over standard 3-drug regimens (3DRs). However, the effect of simplification to 2DRs on HIV-1 reservoir remains to be fully assessed. SETTING: Retrospective analyses of samples of virologically suppressed people living with HIV remaining on the same 3DRs or switching to DTG + 3TC or ATV/r + 3TC 2DRs. METHODS: Whole blood samples were collected at enrollment and after 48 weeks. Total HIV-1 DNA (tDNA) and intact HIV-1 DNA (iDNA) were quantified by droplet digital polymerase chain reaction and intact proviral DNA assay, respectively. Statistical analysis was performed to identify associations among variables, and multiple linear regression was used to analyze potential predictors of tDNA and iDNA changes over time. RESULTS: Forty-seven individuals were switched to DTG + 3TC 2DR (N = 23) and ATV/r + 3TC 2DR (N = 24), while 18 remained on 3DRs. tDNA did not change either in the overall population or in the 3DR and 2DR groups. iDNA decreased significantly in the whole data set and in the overall 3DR and 2DR groups ( P = 0.001, P = 0.039 and P = 0.009, respectively). iDNA, but not tDNA, was inversely correlated with the time of viral suppression ( P = 0.002) and time under antiretroviral therapy ( P = 0.006). Higher nadir CD4 + T-cell counts ( P = 0.001) and lower zenith viral load ( P = 0.02) showed an association with the decrease of iDNA, but not with tDNA. CONCLUSIONS: Both tDNA and iDNA dynamics supported noninferior efficacy of 2DRs over 3DRs. iDNA could be more informative than tDNA in analyzing the dynamics of the HIV-1 reservoir under different treatment strategies.

Long-Term Longitudinal Analysis of Neutralizing Antibody Response to Three Vaccine Doses in a Real-Life Setting of Previously SARS-CoV-2 Infected Healthcare Workers: A Model for Predicting Response to Further Vaccine Doses.

We report the time course of neutralizing antibody (NtAb) response, as measured by authentic virus neutralization, in healthcare workers (HCWs) with a mild or asymptomatic SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection diagnosed at the onset of the pandemic, with no reinfection throughout and after a three-dose schedule of the BNT162b2 mRNA vaccine with an overall follow-up of almost two years since infection. Forty-eight HCWs (median age 47 years, all immunocompetent) were evaluated: 29 (60.4%) were asymptomatic. NtAb serum was titrated at eight subsequent time points: T1 and T2 were after natural infection, T3 on the day of the first vaccine dose, T4 on the day of the second dose, T5, T6, and T7 were between the second and third dose, and T8 followed the third dose by a median of 34 days. NtAb titers at all postvaccination time points (T4 to T8) were significantly higher than all those at prevaccination time points (T1 to T3). The highest NtAb increase was following the first vaccine dose while subsequent doses did not further boost NtAb titers. However, the third vaccine dose appeared to revive waning immunity. NtAb levels were positively correlated at most time points suggesting an important role for immunogenetics.

Efficacy of Licensed Monoclonal Antibodies and Antiviral Agents against the SARS-CoV-2 Omicron Sublineages BA.1 and BA.2.

Newly emerging SARS-CoV-2 variants may escape monoclonal antibodies (mAbs) and antiviral drugs. By using live virus assays, we assessed the ex vivo inhibition of the B.1 wild-type (WT), delta and omicron BA.1 and BA.2 lineages by post-infusion sera from 40 individuals treated with bamlanivimab/etesevimab (BAM/ETE), casirivimab/imdevimab (CAS/IMD), and sotrovimab (SOT) as well as the activity of remdesivir, nirmatrelvir and molnupiravir. mAbs and drug activity were defined as the serum dilution (ID50) and drug concentration (IC50), respectively, showing 50% protection of virus-induced cytopathic effect. All pre-infusion sera were negative for SARS-CoV-2 neutralizing activity. BAM/ETE, CAS/IMD, and SOT showed activity against the WT (ID50 6295 (4355-8075) for BAM/ETE; 18,214 (16,248-21,365) for CAS/IMD; and 456 (265-592) for SOT) and the delta (14,780 (ID50 10,905-21,020) for BAM/ETE; 63,937 (47,211-79,971) for CAS/IMD; and 1103 (843-1334) for SOT). Notably, only SOT was active against BA.1 (ID50 200 (37-233)), whereas BA.2 was neutralized by CAS/IMD (ID50 174 (134-209) ID50) and SOT (ID50 20 (9-31) ID50), but not by BAM/ETE. No significant inter-variant IC50 differences were observed for molnupiravir (1.5 ± 0.1/1.5 ± 0.7/1.0 ± 0.5/0.8 ± 0.01 µM for WT/delta/BA.1/BA.2, respectively), nirmatrelvir (0.05 ± 0.02/0.06 ± 0.01/0.04 ± 0.02/0.04 ± 0.01 µM) or remdesivir (0.08 ± 0.04/0.11 ± 0.08/0.05 ± 0.04/0.08 ± 0.01 µM). Continued evolution of SARS-CoV-2 requires updating the mAbs arsenal, although antivirals have so far remained unaffected.

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro.

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40-80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.

In vitro cross-resistance to doravirine in a panel of HIV-1 clones harbouring multiple NNRTI resistance mutations.

OBJECTIVES: Doravirine is a recently licensed HIV-1 NNRTI with improved efficacy, pharmacokinetics and safety profile compared with efavirenz and limited cross-resistance with rilpivirine and etravirine. In this in vitro study, cross-resistance to doravirine was analysed in a representative panel of NNRTI-resistant clones. METHODS: In vitro phenotypic susceptibility to doravirine was assessed in 10 clinically derived infectious clones with intermediate- to high-level resistance to rilpivirine, etravirine, efavirenz and nevirapine, and in NL4-3 site-directed mutants harbouring K103N, Y181C, M230L or K103N/Y181C NNRTI mutations. RESULTS: Although none of the infectious clones harboured any of the major doravirine resistance-associated mutations (RAMs) included in the IAS-USA reference list, doravirine fold change (FC) values were comparable to or higher than those calculated for other NNRTIs, particularly etravirine and rilpivirine. As expected, single NNRTI mutations K103N and Y181C did not impair doravirine susceptibility (FC 1.4 and 1.8, respectively), while reduced activity was observed with the single M230L or double K103N/Y181C mutations (FC 7.6 and 4.9, respectively). Median FC values increased significantly with increasing numbers of NNRTI RAMs (P = 0.005) and were >10 in 4/4 and 1/4 clones harbouring four and three NNRTI RAMs, respectively. FC values correlated well with predicted susceptibility as inferred by Stanford HIV Drug Resistance Database (HIVdb) and ANRS algorithms (both P < 0.001). CONCLUSIONS: Substantial cross-resistance to doravirine was detected in NNRTI-resistant viruses harbouring complex mutational patterns, even in the absence of major IAS-USA doravirine RAMs. Therefore, based on the simple IAS-USA reference list, doravirine resistance may be underestimated in viruses harbouring multiple NNRTI mutations.

In vitro susceptibility to fostemsavir is not affected by long-term exposure to antiviral therapy in MDR HIV-1-infected patients.

OBJECTIVES: Fostemsavir is the prodrug of the HIV-1 attachment inhibitor temsavir and is currently under clinical assessment in heavily treatment-experienced patients with limited therapeutic options. We evaluated the genotypic and phenotypic susceptibility to temsavir in a panel of samples collected from patients harbouring MDR strains enrolled in the Italian PRESTIGIO Registry. METHODS: Plasma samples from 24 patients were used for HIV-1 gp120 sequencing, while viral tropism and susceptibility to temsavir were assessed through a homemade phenotypic assay with pseudotyped viruses expressing patient-derived Env protein. RESULTS: Of the 24 patients enrolled, 18 (75%) were male, median (IQR) age was 55 years (52-61), time since HIV-1 diagnosis was 27 years (24-30), time on ART was 26 years (23-27) and 11 (46%) had a previous AIDS diagnosis. Exposure to entry inhibitors (maraviroc and/or enfuvirtide) had occurred in 19 (79%) patients. Among 23/24 gp120 sequences obtained, temsavir resistance-associated mutations (RAMs) were detected in three cases (two M426L and one S375N). Pseudotyped viruses were obtained from 23/24 samples and viral tropism was CXCR4-tropic, CCR5-tropic and dual/mixed-tropic in six, nine and eight cases, respectively. Phenotypic susceptibility to temsavir was comparable to the reference WT viruses NL4-3 and AD8 in all samples, irrespective of RAMs. Viral tropism and exposure to entry inhibitors did not impact temsavir susceptibility. CONCLUSIONS: These data support the use of fostemsavir as a valuable therapy option in patients harbouring MDR virus. The role of laboratory testing in optimal screening of patients eligible for fostemsavir treatment remains to be investigated.

Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication.

Practical cell-based assays can accelerate anti-Zika (ZIKV) and anti-dengue (DENV) virus drug discovery. We developed an immunodetection assay (IA), using a pan-flaviviral monoclonal antibody recognizing a conserved envelope domain. The final protocol includes a direct virus yield reduction assay (YRA) carried out in the human Huh7 cell line, followed by transfer of the supernatant to a secondary Huh7 culture to characterize late antiviral effects. Sofosbuvir and ribavirin were used to validate the assay, while celgosivir was used to evaluate the ability to discriminate between early and late antiviral activity. In the direct YRA, at 100, 50, and 25 TCID50, sofosbuvir IC50 values were 5.0 ± 1.5, 2.7 ± 0.5, 2.5 ± 1.1 µM against ZIKV and 16.6 ± 2.8, 4.6 ± 1.4, 2.6 ± 2.2 µM against DENV; ribavirin IC50 values were 6.8 ± 4.0, 3.8 ± 0.6, 4.5 ± 1.4 µM against ZIKV and 17.3 ± 4.6, 7.6 ± 1.2, 4.1 ± 2.3 µM against DENV. Sofosbuvir and ribavirin IC50 values determined in the secondary YRA were reproducible and comparable with those obtained by direct YRA and plaque reduction assay (PRA). In agreement with the proposed mechanism of late action, celgosivir was active against DENV only in the secondary YRA (IC50 11.0 ± 1.0 µM) and in PRA (IC50 10.1 ± 1.1 µM). The assay format overcomes relevant limitations of the gold standard PRA, allowing concurrent analysis of candidate antiviral compounds against different viruses and providing preliminary information about early versus late antiviral activity.

Evaluation of sofosbuvir activity and resistance profile against West Nile virus in vitro.

Sofosbuvir, a licensed nucleotide analog targeting hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), has been recently evaluated as a broad anti-Flavivirus lead candidate revealing activity against Zika and Dengue viruses both in vitro and in animal models. In this study, the in vitro antiviral activity of sofosbuvir against West Nile virus (WNV) was determined by plaque assay (PA) and Immunodetection Assay (IA) in human cell lines and by enzymatic RdRp assay. By PA, the sofosbuvir half-maximal inhibitory concentration (IC50) was 1.2 ± 0.3 µM in Huh-7, 5.3 ± 0.9 µM in U87, 7.8 ± 2.5 µM in LN-18 and 63.4 ± 14.1 µM in A549 cells. By IA, anti-WNV activity was confirmed in both hepatic (Huh-7, 1.7 ± 0.5 µM) and neuronal (U87, 7.3 ± 2.0 µM) cell types. Sofosbuvir was confirmed to inhibit the purified WNV RdRp (IC50 11.1 ± 4.6 µM). In vitro resistance selection experiments were performed by propagating WNV in the Huh-7 cell line with two-fold increasing concentrations of sofosbuvir. At 80 µM, a significantly longer time for viral breakthrough was observed compared with lower concentrations (18 vs. 7-9 days post infection; p = 0.029), along with the detection of the S604T mutation, corresponding to the well-known S282T substitution in the motif B of HCV NS5B, which confers resistance to sofosbuvir. Molecular docking experiments confirmed that the S604T mutation within the catalytic site of RdRp affected the binding mode of sofosbuvir. To our knowledge, this is the first report of the antiviral activity of sofosbuvir against WNV as well as of selection of mutants in vitro.