Revamped role for approved drug: integrative computational and biophysical analysis of saquinavir's peptidyl arginine deiminase 4 inhibition for rheumatoid arthritis.

The pursuit of novel therapeutics is a complex and resource-intensive endeavor marked by significant challenges, including high costs and low success rates. In response, drug repositioning strategies leverage existing FDA-approved compounds to predict their efficacy across diverse diseases. Peptidyl arginine deiminase 4 (PAD4) plays a pivotal role in protein citrullination, a process implicated in the autoimmune pathogenesis of rheumatoid arthritis (RA). Targeting PAD4 has thus emerged as a promising therapeutic approach. This study employs computational and enzyme inhibition strategies to identify potential PAD4-targeting compounds from a library of FDA-approved drugs. In silico docking analyses validated the binding interactions and orientations of screened compounds within PAD4's active site, with key residues such as ASP350, HIS471, ASP473, and CYS645 participating in crucial hydrogen bonding and van der Waals interactions. Molecular dynamics simulations further assessed the stability of top compounds exhibiting high binding affinities. Among these compounds, Saquinavir (SQV) emerged as a potent PAD4 inhibitor, demonstrating competitive inhibition with a low IC50 value of 1.21 ± 0.04 µM. In vitro assays, including enzyme kinetics and biophysical analyses, highlighted significant changes in PAD4 conformation upon SQV binding, as confirmed by circular dichroism spectroscopy. SQV induced localized alterations in PAD4 structure, effectively occupying the catalytic pocket and inhibiting enzymatic activity. These findings underscore SQV's potential as a therapeutic candidate for RA through PAD4 inhibition. Further validation through in vitro and in vivo studies is essential to confirm SQV's therapeutic benefits in autoimmune diseases associated with dysregulated citrullination.

The Use of the Protease Inhibitor, Saquinavir, to Treat Anal Cancer Spheroids Derived From Human Papillomavirus Transgenic Mice.

BACKGROUND: Anal cancer is associated with high-risk human papillomavirus infection and oncoprotein expression. We have identified several protease inhibitors, used to treat HIV, that decrease oncogene expression. OBJECTIVE: The aim of this project is to determine whether saquinavir, a protease inhibitor, results in a treatment response in anal cancer spheroids. DESIGN: K14E6/E7 transgenic mice (n = 5), which express human papillomavirus 16 oncoproteins E6 and E7 in their epithelium, were treated topically at the anus with a carcinogen, 7,12-dimethylbenz[a]anthracene, to promote anal tumor growth. Tumors were excised and digested, and cells were plated. The tumor cells form 3D multicellular aggregates known as spheroids. SETTINGS: This study was performed in an American Association for Accreditation of Laboratory Animal Care-approved facility. INTERVENTIONS: Spheroids were placed in treatment groups: no treatment, vehicle (dimethyl sulfoxide), and 15 µM saquinavir. Spheroids were imaged immediately pretreatment and 24 hours posttreatment. MAIN OUTCOME MEASURES: Spheroid diameters were measured using ImageJ and mean percent reduction was calculated for each spheroid to determine treatment effect on spheroid growth. Analysis of variance using pairwise comparisons was performed with Fisher protected least significant difference tests. RESULTS: The no-treatment (n = 119 spheroids) and vehicle (n = 126 spheroids) groups demonstrated an increase in spheroid diameter during the treatment period. In contrast, spheroids treated with saquinavir (n = 151 spheroids) demonstrated a statistically significant percent reduction compared to the no-treatment ( p < 0.0001) and vehicle ( p = 0.002) groups. LIMITATIONS: A limitation of these data is that some human error is likely present given that images were analyzed by 3 different scientists. CONCLUSIONS: Saquinavir leads to a statistically significant percent reduction in mice anal tumor spheroid growth ex vivo compared to control groups. Protease inhibitor therapy may be an effective treatment or adjuvant therapy to the Nigro protocol to promote anal cancer tumor regression. See Video Abstract at http://links.lww.com/DCR/C82 . EL USO DEL INHIBIDOR DE LA PROTEASA, SAQUINAVIR, PARA TRATAR LOS ESFEROIDES DEL CNCER ANAL DERIVADOS DE RATONES TRANSGNICOS PARA EL VPH: ANTECEDENTES:El cáncer anal está asociado con la infección por el virus del papiloma humano de alto riesgo y la expresión de oncoproteínas. Hemos identificado varios inhibidores de la proteasa, utilizados para tratar el VIH, que disminuyen la expresión del oncogén.OBJETIVO:El objetivo de este proyecto es determinar si los esferoides de cáncer anal responden al tratamiento con inhibidor de la proteasa, Saquinavir.DISEÑO:Ratones transgénicos K14E6/E7 (n = 5), que expresan las oncoproteínas E6 y E7 del VPH16 en su epitelio, fueron tratados tópicamente en el ano con carcinógeno, 7,12 dimetilbenz[a]antraceno, para promover el crecimiento del tumor anal. Los tumores se extirparon y digirieron, y las células se sembraron en placas. Las células tumorales forman agregados multicelulares tridimensionales, conocidos como esferoides.ESCENARIO:Este estudio se realizó en un centro aprobado por la Asociación Estadounidense para la Acreditación de Cuidado de Animales de Laboratorio.INTERVENCIONES:Se colocaron esferoides en grupos de tratamiento: sin tratamiento, vehículo (sulfóxido de dimetilo) y saquinavir 15 µM. Se tomaron imágenes de los esferoides inmediatamente antes del tratamiento y 24 horas después del tratamiento.PRINCIPALES MEDIDAS DE RESULTADO:Los diámetros de los esferoides se midieron con ImageJ y se calculó el porcentaje medio de reducción de cada esferoide para determinar el efecto del tratamiento sobre el crecimiento de los esferoides. El análisis de varianza mediante comparaciones por pares se realizó con las pruebas de diferencia mínima significativa protegida de Fisher.RESULTADOS:Los grupos sin tratamiento (n =119 esferoides) y vehículo (n=126 esferoides) demostraron un aumento en el diámetro del esferoide durante el período de tratamiento. Por el contrario, los esferoides tratados con saquinavir (n =151 esferoides) demostraron una reducción porcentual estadísticamente significativa en comparación con los grupos sin tratamiento ( p < 0,0001) y con vehículo (p = 0,002).LIMITACIONES:una limitación de estos datos es que es probable que haya algún error humano dado que las imágenes fueron analizadas por tres científicos diferentes.CONCLUSIONES:Saquinavir conduce a una reducción porcentual estadísticamente significativa en el crecimiento de esferoides de tumores anales en ratones ex-vivo en comparación con los grupos de control. La terapia con inhibidores de la proteasa puede ser un tratamiento eficaz o una terapia adyuvante del protocolo Nigro para promover la regresión del tumor del cáncer anal. Consulte Video Resumen en http://links.lww.com/DCR/C82 . (Traducción-Dr. Felipe Bellolio ).

Saquinavir potentiates itraconazole's antifungal activity against multidrug-resistant Candida auris in vitro andin vivo.

Candida species are highly opportunistic yeasts that are responsible for serious invasive fungal infections among immunocompromised patients worldwide. Due to the increase in drug resistance and incidence of infections, there is an urgent need to develop new antifungals and to identify co-drugs that can sensitize drug-resistant Candida to antifungals. The objective of this study was to assess the effect of saquinavir on the activity of azole antifungals against C. auris. The in vitro interaction of saquinavir and three azole antifungals (itraconazole, voriconazole, and fluconazole) was evaluated against a panel of C. auris isolates. The itraconazole/saquinavir combination exhibited a synergistic (SYN) relationship against all C. auris isolates tested with the fractional inhibitory concentration index ranging from 0.03 to 0.27. Moreover, a time-kill kinetics assay revealed that saquinavir restored the itraconazole's fungistatic activity against C. auris. Furthermore, saquinavir restored itraconazole's antifungal activity against other clinically important Candida species. The mechanistic investigation indicated that saquinavir significantly inhibited efflux pumps, glucose utilization, and ATP synthesis in Candida. Finally, a murine model of C. auris infection was used to evaluate the efficacy of the itraconazole/saquinavir combination in the presence of ritonavir (as a pharmacokinetic enhancer). The combination significantly reduced the fungal burden in the kidneys by 0.93-log10 colony-forming units (88%) compared to itraconazole alone. This study identified that saquinavir exhibits a potent SYN relationship in combination with itraconazole against Candida species, which warrants further consideration.

Candida auris is a multi-drug resistant fungal pathogen with limited treatment options. In this study, we identified that the antiviral drug, saquinavir, is capable of synergizing and restoring the activity of antifungals against C. auris.

Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system.

A genetic selection system for activity of HIV protease is described that is based on a synthetic substrate constructed as a modified AraC regulatory protein that when cleaved stimulate l-arabinose metabolism in an Escherichia coli araC strain. Growth stimulation on selective plates was shown to depend on active HIV protease and the scissile bond in the substrate. In addition, the growth of cells correlated well with the established cleavage efficiency of the sites in the viral polyprotein, Gag, when these sites were individually introduced into the synthetic substrate of the selection system. Plasmids encoding protease variants selected based on stimulation of cell growth in the presence of saquinavir or cleavage of a site not cleaved by wild-type protease, were indistinguishable with respect to both phenotypes. Also, both groups of selected plasmids encoded side chain substitutions known from clinical isolates or displayed different side chain substitutions but at identical positions. One highly frequent side chain substitution, E34V, not regarded as a major drug resistance substitution was found in variants obtained under both selective conditions and is suggested to improve protease processing of the synthetic substrate. This substitution is away from the substrate-binding cavity and together with other substitutions in the selected reading frames supports the previous suggestion of a substrate-binding site extended from the active site binding pocket itself.

HIV protease inhibitor saquinavir inhibits toll-like receptor 4 activation by targeting receptor dimerization.

OBJECTIVE: Toll-like receptor 4 (TLR4) is crucial in induction of innate immune response through recognition of invading pathogens or endogenous alarming molecules. Ligand-triggered dimerization of TLR4 is essential for the activation of NF-κB and IRF3 through MyD88- or TRIF-dependent pathways. Saquinavir (SQV), an FDA-approved HIV protease inhibitor, has been shown to attenuate the activation of NF-κB induced by HMGB1 by blocking TLR4-MyD88 association in proteasome independent pathway. This study aims to define whether SQV is an HMGB1-specific and MyD88-dependent TLR4 signaling inhibitor and which precise signaling element of TLR4 is targeted by SQV. MATERIALS AND METHODS: PMA differentiated human THP-1 macrophages or reconstituted HEK293 cells were pretreated with SQV before stimulated by different TLR agonists. TNF-α level was evaluated through ELISA assay. NF-κB activation was analyzed using NF-κB SEAP reporting system. The levels of MyD88/TRIF pathways-related factors were examined by immunoblot. TLR4 endocytosis was assessed by immunocytochemistry. TLR4 dimerization was determined using immunoprecipitation between different tagged TLR4 and an in silico molecular docking experiment was performed to explore the possible binding site of SQV on its target. RESULTS: Our data showed that SQV suppresses both MyD88- and TRIF-dependent pathways in response to lipopolysaccharide (LPS), a critical sepsis inducer and TLR4 agonist, leading to downregulation of NF-κB and IRF3. SQV did not suppress MyD88-dependent pathway triggered by TLR1/2 agonist Pam3csk4. In the only TRIF-dependent pathway, SQV did not alleviate IRF3 phosphorylation induced by TLR3 agonist Poly(I:C). Furthermore, dimerization of TLR4 following LPS or HMGB1 stimulation was decreased by SQV. CONCLUSION: We concluded that TLR4 receptor complex is one of the mammalian targets of SQV, and TLR4-mediated immune responses and consequent risk for uncontrolled inflammation could be modulated by FDA-approved drug SQV.

Liposomal Delivery of Saquinavir to Macrophages Overcomes Cathepsin Blockade by Mycobacterium tuberculosis and Helps Control the Phagosomal Replicative Niches.

Mycobacterium tuberculosis is able to establish a chronic colonization of lung macrophages in a controlled replication manner, giving rise to a so-called latent infection. Conversely, when intracellular bacteria undergo actively uncontrolled replication rates, they provide the switch for the active infection called tuberculosis to occur. Our group found that the pathogen is able to manipulate the activity of endolysosomal enzymes, cathepsins, directly at the level of gene expression or indirectly by regulating their natural inhibitors, cystatins. To provide evidence for the crucial role of cathepsin manipulation for the success of tuberculosis bacilli in their intracellular survival, we used liposomal delivery of saquinavir. This protease inhibitor was previously found to be able to increase cathepsin proteolytic activity, overcoming the pathogen induced blockade. In this study, we demonstrate that incorporation in liposomes was able to increase the efficiency of saquinavir internalization in macrophages, reducing cytotoxicity at higher concentrations. Consequently, our results show a significant impact on the intracellular killing not only to reference and clinical strains susceptible to current antibiotic therapy but also to multidrug- and extensively drug-resistant (XDR) Mtb strains. Altogether, this indicates the manipulation of cathepsins as a fine-tuning strategy used by the pathogen to survive and replicate in host cells.

Repurposing Alone and in Combination of the Antiviral Saquinavir with 5-Fluorouracil in Prostate and Lung Cancer Cells.

Prostate and lung cancers are among the most common cancer types, and they still need more therapeutics. For this purpose, saquinavir (SAQ) was tested alone and in combination with 5-fluorouracil (5-FU). PC-3 and A549 cells were exposed to increasing concentrations of both drugs alone or in combination, with simultaneous or sequential administration. Cell viability was obtained using the MTT assay and synergism values using CompuSyn software. Results showed that SAQ was the more cytotoxic of both drugs in PC-3 cells, while 5-FU was the most cytotoxic in A549 cells. When these drugs were used in combination, the more synergistic combination in PC-3 cells was the IC50 of SAQ with various concentrations of 5-FU, particularly when 5-FU was only applied 24 h later. Meanwhile for A549 the most promising combination was 5-FU with delayed SAQ, but with a weaker effect than all combinations demonstrated in PC-3 cells. These results demonstrate that SAQ could be used as a new repurposed drug for the treatment of prostate cancer and this treatment potential could be even greater if SAQ is combined with the anticancer drug 5-FU, while for lung cancer it is not as efficient and, therefore, not of as much interest.

Discovery of Novel HIV Protease Inhibitors Using Modern Computational Techniques.

The human immunodeficiency virus type 1 (HIV-1) has continued to be a global concern. With the new HIV incidence, the emergence of multi-drug resistance and the untoward side effects of currently used anti-HIV drugs, there is an urgent need to discover more efficient anti-HIV drugs. Modern computational tools have played vital roles in facilitating the drug discovery process. This research focuses on a pharmacophore-based similarity search to screen 111,566,735 unique compounds in the PubChem database to discover novel HIV-1 protease inhibitors (PIs). We used an in silico approach involving a 3D-similarity search, physicochemical and ADMET evaluations, HIV protease-inhibitor prediction (IC50/percent inhibition), rigid receptor-molecular docking studies, binding free energy calculations and molecular dynamics (MD) simulations. The 10 FDA-approved HIV PIs (saquinavir, lopinavir, ritonavir, amprenavir, fosamprenavir, atazanavir, nelfinavir, darunavir, tipranavir and indinavir) were used as reference. The in silico analysis revealed that fourteen out of the twenty-eight selected optimized hit molecules were within the acceptable range of all the parameters investigated. The hit molecules demonstrated significant binding affinity to the HIV protease (PR) when compared to the reference drugs. The important amino acid residues involved in hydrogen bonding and п-п stacked interactions include ASP25, GLY27, ASP29, ASP30 and ILE50. These interactions help to stabilize the optimized hit molecules in the active binding site of the HIV-1 PR (PDB ID: 2Q5K). HPS/002 and HPS/004 have been found to be most promising in terms of IC50/percent inhibition (90.15%) of HIV-1 PR, in addition to their drug metabolism and safety profile. These hit candidates should be investigated further as possible HIV-1 PIs with improved efficacy and low toxicity through in vitro experiments and clinical trial investigations.

Polycaprolactone and polycaprolactone triol blends to obtain a stable liquid nanotechnological formulation: synthesis, characterization and in vitro - in vivo taste masking evaluation.

The use of polymeric blends is a potential strategy to obtain novel nanotechnological formulations aiming at drug delivery systems. Saquinavir, an antiretroviral drug, was chosen as a model drug for the development of new stable liquid formulations with unpleasant taste masking properties. Three formulations containing different polymeric ratios (1:3, 1:1 and 3:1) were prepared and properly characterized by particle size distribution, zeta potential, pH, drug content and encapsulation efficiency measurements. The stability was verified by monitoring the zeta potential, particle size distribution, polydispersity index and drug content by 90 days. The light backscattering analysis was used to early identify possible phenomena of instability in the formulations. The in vitro drug release and saquinavir cytotoxicity were evaluated. The in vitro and in vivo taste masking properties were studied using an electronic tongue and a human sensory panel. All formulations presented nanometric sizes around 200 nm and encapsulation efficiency above 99%. The parameters evaluated for stability remained constant throughout 90 days. The in vitro tests showed a controlled drug release and absence of toxic effects on human T lymphocytes. The electronic tongue experiment showed taste differences for all formulations in comparison to drug solutions, with a more pronounced difference for the formulation with higher polycaprolactone content (3:1). This formulation was chosen for in vivo sensory panel evaluation which results corroborated the electronic tongue experiments. In conclusion, the polymer blend nanoformulation developed herein showed the promising application to incorporate drugs aiming at pharmaceutical taste-masking properties.

Anti-HIV and Anti-Hepatitis C Virus Drugs Inhibit P-Glycoprotein Efflux Activity in Caco-2 Cells and Precision-Cut Rat and Human Intestinal Slices.

P-glycoprotein (ABCB1), an ATP-binding-cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions (DDIs). ABCB1 has been suggested to interact with many antivirals used to treat HIV and/or chronic hepatitis C virus (HCV) infections. Using bidirectional transport experiments in Caco-2 cells and a recently established ex vivo model of accumulation in precision-cut intestinal slices (PCIS) prepared from rat ileum or human jejunum, we evaluated the potential of anti-HIV and anti-HCV antivirals to inhibit intestinal ABCB1. Lopinavir, ritonavir, saquinavir, atazanavir, maraviroc, ledipasvir, and daclatasvir inhibited the efflux of a model ABCB1 substrate, rhodamine 123 (RHD123), in Caco-2 cells and rat-derived PCIS. Lopinavir, ritonavir, saquinavir, and atazanavir also significantly inhibited RHD123 efflux in human-derived PCIS, while possible interindividual variability was observed in the inhibition of intestinal ABCB1 by maraviroc, ledipasvir, and daclatasvir. Abacavir, zidovudine, tenofovir disoproxil fumarate, etravirine, and rilpivirine did not inhibit intestinal ABCB1. In conclusion, using recently established ex vivo methods for measuring drug accumulation in rat- and human-derived PCIS, we have demonstrated that some antivirals have a high potential for DDIs on intestinal ABCB1. Our data help clarify the molecular mechanisms responsible for reported increases in the bioavailability of ABCB1 substrates, including antivirals and drugs prescribed to treat comorbidity. These results could help guide the selection of combination pharmacotherapies and/or suitable dosing schemes for patients infected with HIV and/or HCV.

The inhibition effect of garlic-derived compounds on human immunodeficiency virus type 1 and saquinavir.

Garlic has been used as a traditional medicine to treat various diseases. Garlic reduces the risk of some diseases. This protective effect is due to the organosulfur compounds of garlic. The aim of this study was to investigate the inhibition effects of garlic-derived compounds on human immunodeficiency virus type 1 (HIV-1) and as the most important anti-HIV-1 medicine. The activation of saquinavir is believed to be the principal mechanism behind the protective effects of HIV-1. Our theoretical calculations are performed for blood phase by using the density functional theory for the main compounds of garlic. The chemical activity and solubility of ajoene and the mainly derived compounds of garlic as theoretical calculations are important for the medical research comparing with the other compounds of the garlic. The theoretical calculations have helped us to determine which active ingredient of the garlic having inhibition effects on HIV-1 and saquinavir.

High-mobility group box 1 protein is involved in the protective effect of Saquinavir on ventilation-induced lung injury in mice.

Saquinavir (SQV) is the first FDA approved HIV protease inhibitor. Previous studies showed that SQV can limit Toll-like receptor-4 (TLR4)-mediated inflammatory pathway and nuclear factor-κB (NF-κB) activation, thereby playing a protective role in many kinds of diseases. High-mobility group box 1 (HMGB1) has been identified as an inflammatory mediator and it might express its toxicity in a short period of time in ventilator-induced lung injury (VILI). In this study, C57BL/6 mice were randomly divided into four groups (n = 10): control group and control with SQV group (Con + SQV) were spontaneous breath. HTV group (HTV) received high tidal volume ventilation (HTV) for 4 h. HTV with SQV group (HTV + SQV) were pretreated with 5 mg/kg of SQV for 7 days before HTV. Mice were sacrificed after 4 h of HTV. Lung wet/dry weight (W/D) ratio, alveolar-capillary permeability to Evans blue albumin (EBA), cell counts, total proteins in bronchoalveolar lavage fluid (BALF), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) level in BALF and lung tissue, and lung histopathology were examined. Our results showed that HTV caused significant lung injury and NF-κB activation, which was correlated with the increase of TNF-α and IL-6 levels in BALF and plasma. SQV pretreatment significantly attenuated pulmonary inflammatory injury, as well as NF-κB activation. These findings indicate that the protective effect of SQV may be associated with the inhibition of NF-κB activation and HMGB1 expression in mice.

Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage.

HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine.

Defective hydrophobic sliding mechanism and active site expansion in HIV-1 protease drug resistant variant Gly48Thr/Leu89Met: mechanisms for the loss of saquinavir binding potency.

HIV drug resistance continues to emerge; consequently, there is an urgent need to develop next generation antiretroviral therapeutics.1 Here we report on the structural and kinetic effects of an HIV protease drug resistant variant with the double mutations Gly48Thr and Leu89Met (PRG48T/L89M), without the stabilizing mutations Gln7Lys, Leu33Ile, and Leu63Ile. Kinetic analyses reveal that PRG48T/L89M and PRWT share nearly identical Michaelis-Menten parameters; however, PRG48T/L89M exhibits weaker binding for IDV (41-fold), SQV (18-fold), APV (15-fold), and NFV (9-fold) relative to PRWT. A 1.9 Å resolution crystal structure was solved for PRG48T/L89M bound with saquinavir (PRG48T/L89M-SQV) and compared to the crystal structure of PRWT bound with saquinavir (PRWT-SQV). PRG48T/L89M-SQV has an enlarged active site resulting in the loss of a hydrogen bond in the S3 subsite from Gly48 to P3 of SQV, as well as less favorable hydrophobic packing interactions between P1 Phe of SQV and the S1 subsite. PRG48T/L89M-SQV assumes a more open conformation relative to PRWT-SQV, as illustrated by the downward displacement of the fulcrum and elbows and weaker interatomic flap interactions. We also show that the Leu89Met mutation disrupts the hydrophobic sliding mechanism by causing a redistribution of van der Waals interactions in the hydrophobic core in PRG48T/L89M-SQV. Our mechanism for PRG48T/L89M-SQV drug resistance proposes that a defective hydrophobic sliding mechanism results in modified conformational dynamics of the protease. As a consequence, the protease is unable to achieve a fully closed conformation that results in an expanded active site and weaker inhibitor binding.

Evaluation of chitosan nanoformulations as potent anti-HIV therapeutic systems.

BACKGROUND: Antiretroviral Therapy (ART) is currently the major therapeutic intervention in the treatment of AIDS. ART, however, is severely limited due to poor availability, high cytotoxicity, and enhanced metabolism and clearance of the drug molecules by the renal system. The use of nanocarriers encapsulating the anti-retroviral drugs may provide a solution to the aforementioned problems. Importantly, the application of mildly immunogenic polymeric carrier confers the advantage of making the nanoparticles more visible to the immune system leading to their efficient uptake by the phagocytes. METHODS: The saquinavir-loaded chitosan nanoparticles were characterized by transmission electron microscopy and differential scanning calorimetry and analyzed for the encapsulation efficiency, swelling characteristics, particle size properties, and the zeta potential. Furthermore, cellular uptake of the chitosan nanocarriers was evaluated using confocal microscopy and Flow cytometry. The antiviral efficacy was quantified using viral infection of the target cells. RESULTS: Using novel chitosan carriers loaded with saquinavir, a protease inhibitor, we demonstrate a drug encapsulation efficiency of 75% and cell targeting efficiency greater than 92%. As compared to the soluble drug control, the saquinavir-loaded chitosan carriers caused superior control of the viral proliferation as measured by using two different viral strains, NL4-3 and Indie-C1, and two different target T-cells, Jurkat and CEM-CCR5. CONCLUSION: Chitosan nanoparticles loaded with saquinavir were characterized and they demonstrated superior drug loading potential with greater cell targeting efficiency leading to efficient control of the viral proliferation in target T-cells. GENERAL SIGNIFICANCE: Our data ascertain the potential of chitosan nanocarriers as novel vehicles for HIV-1 therapeutics.

Development and characterization of folate anchored Saquinavir entrapped PLGA nanoparticles for anti-tumor activity.

OBJECTIVE: Saquinavir (SQV) is a US-FDA approved HIV protease inhibitor (HPI) for HIV cure. The purpose of the present investigation was to develop and characterize the anticancer potential of the SQV-loaded folic acid (FA) conjugated PEGylated and non-PEGylated poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) (SQV-Fol-PEG-PLGA and SQV-Fol-PLGA) employing PC-3 (human prostate) and MCF-7 (human breast) cancer cell lines. MATERIALS AND METHODS: Developed NPs were characterized by IR, NMR, DSC, XRD, size, charge and further tested for drug loading and cellular uptake properties. RESULT: The entrapment efficiency was found to be 56 ± 0.60 and 58 ± 0.80 w/v for SQV-Fol-PEG-PLGA and SQV-PLGA NPs, respectively. The obtained results of SQV-Fol-PEG-PLGA showed enhanced cytotoxicity and cellular uptake and were most preferentially taken up by the cancerous cells via folate receptor-mediated endocytosis (RME) mechanism. At 260 µM concentration, SQV-PLGA NPs and SQV-Fol-PEG-PLGA NPs showed 20%, 20% and 23% cell growth inhibition in PC-3 cells, respectively whereas in MCF-7 cells it was 12%, 15% and 14% cell growth inhibition, respectively. CONCLUSIONS: Developed targeted SQV-Fol-PEG-PLGA NPs were superior anticancer potential as compared to non-targeted SQV-PLGA NPs. Thus, these targeted NPs provide another option for anticancer drug delivery scientists.

[Effect and mechanism of endoplasmic reticulum stress on cisplatin resistance in ovarian carcinoma].

OBJECTIVE: The study intended to investigate the effect and mechanism of endoplasmic reticulum stress on cisplatin resistance in ovarian carcinoma. METHODS: RT-PCR and Western blot were used to test the expression of mTOR and Beclin1 mRNA and protein in ovarian cancer SKOV3 cells after saquinavir induction. MTT assay was used to analyze the influence of saquinavir on cisplatin sensitivity in SKOV3 cells. RESULTS: The IC50 of SKOV3 cells was (5.490 ± 1.148) µg/ml. After induced by Saquinavair 10 µmol/L and 20 µmol/L, the IC50 of SKOV3 cells was increased to (11.199 ± 0.984) µg/ml and (14.906 ± 2.015) µg/ml, respectively. It suggested that the sensitivity of ovarian cancer cells to cisplatin was decreased significantly (P = 0.001). The expression of mTOR and Beclin1 mRNA and protein was significantly different among the five groups: the (Saquinavair+DDP) group of, Saquinavair group, LY294002 group, DDP group and control group (P < 0.001) . The expressions of mTOR and Beclin1 mRNA were highest in the (Saquinavair+DDP) group, 0.684 ± 0.072 and 0.647 ± 0.047, respectively; Secondly, the Saquinavair group, 0.577 ± 0.016 and 0.565 ± 0.037, respectively. The expressions of mTOR and Beclin1 proteins were also highest in the (Saquinavair+DDP) group, 0.624 ± 0.058 and 0.924 ± 0.033, respectively, followed by the Saquinavair group, 0.544 ± 0.019 and 0.712 ± 0.024. 3-MA inhibited the autophagy and restored cisplatin sensitivity in the SKOV3 cells after Saquinavir induced ER stress (P < 0.001). CONCLUSIONS: Saquinavir can effectively induce endoplasmic reticulum stress in SKOV3 cells. Endoplasmic reticulum stress can decrease the sensitivity to cisplatin in SKOV3 cells. The mechanism of the decrease of sensitivity to cisplatin in SKOV3 cells may be that ERS regulates cell autophagy through the mTOR and Beclin1 pathways. ERS of tumor cells and autophagy may become a new target to improve the therapeutic effect of chemotherapy and to reverse the drug resistance in tumor treatment.

HIV treatments have malaria gametocyte killing and transmission blocking activity.

BACKGROUND: Millions of individuals being treated for human immunodeficiency virus (HIV) live in malaria-endemic areas, but the effects of these treatments on malaria transmission are unknown. While drugs like HIV protease inhibitors (PIs) and trimethoprim-sulfamethoxazole (TMP-SMX) have known activity against parasites during liver or asexual blood stages, their effects on transmission stages require further study. METHODS: The HIV PIs lopinavir and saquinavir, the nonnucleoside reverse-transcriptase inhibitor nevirapine, and the antibiotic TMP-SMX were assessed for activity against Plasmodium falciparum transmission stages. The alamarBlue assay was used to determine the effects of drugs on gametocyte viability, and exflagellation was assessed to determine the effects of drugs on gametocyte maturation. The effects of drug on transmission were assessed by calculating the mosquito oocyst count as a marker for infectivity, using standard membrane feeding assays. RESULTS: Lopinavir and saquinavir have gametocytocidal and transmission blocking activities at or approaching clinically relevant treatment levels, while nevirapine does not. TMP-SMX is not gametocytocidal, but at prophylactic levels it blocks transmission. CONCLUSIONS: Specific HIV treatments have gametocyte killing and transmission-blocking effects. Clinical studies are warranted to evaluate these findings and their potential impact on eradication efforts.

Enhanced antifungal activity of posaconazole against Candida auris by HIV protease inhibitors, atazanavir and saquinavir.

The increasing incidence and dissemination of multidrug-resistant Candida auris represents a serious global threat. The emergence of pan-resistant C. auris exhibiting resistance to all three classes of antifungals magnifies the need for novel therapeutic interventions. We identified that two HIV protease inhibitors, atazanavir and saquinavir, in combination with posaconazole exhibited potent activity against C. auris in vitro and in vivo. Both atazanavir and saquinavir exhibited a remarkable synergistic activity with posaconazole against all tested C. auris isolates and other medically important Candida species. In a time-kill assay, both drugs restored the fungistatic activity of posaconazole, resulting in reduction of 5 and 5.6 log10, respectively. Furthermore, in contrast to the individual drugs, the two combinations effectively inhibited the biofilm formation of C. auris by 66.2 and 81.2%, respectively. Finally, the efficacy of the two combinations were tested in a mouse model of C. auris infection. The atazanavir/posaconazole and saquinavir/posaconazole combinations significantly reduced the C. auris burden in mice kidneys by 2.04- (99.1%) and 1.44-log10 (96.4%) colony forming unit, respectively. Altogether, these results suggest that the combination of posaconazole with the HIV protease inhibitors warrants further investigation as a new therapeutic regimen for the treatment of C. auris infections.

The SARS-CoV-2 Mpro Dimer-Based Screening System: A Synthetic Biology Tool for Identifying Compounds with Dimerization Inhibitory Potential.

The COVID-19 endemic remains a global concern. The search for effective antiviral candidates is still needed to reduce disease risk. However, the availability of high biosafety level laboratory facilities for drug screening is limited in number. To address this issue, a screening system that could be utilized at lower biosafety levels remains essential. This study aimed to develop a novel SARS-CoV-2 main protease (Mpro) dimer-based screening system (DBSS) utilizing synthetic biology in Escherichia coli BL21(DE3). We linked the SARS-CoV-2 Mpro with the DNA-binding domain of AraC regulatory protein, which regulates the reporter gene expression. Protein modeling and molecular docking showed that saquinavir could bind to AraC-Mpro both in its monomer and dimer forms. The constructed DBSS assay indicated the screening system could detect saquinavir inhibitory activity at a concentration range of 4-10 µg/mL compared to the untreated control (P ≤ 0.05). The Vero E6 cell assay validated the DBSS result that saquinavir at 4-10 µg/mL exhibited antiviral activity against SARS-CoV-2. Our DBSS could be used for preliminary screening of numerous drug candidates that possess a dimerization inhibitor activity of SARS-CoV-2 Mpro and also minimize the use of a high biosafety level laboratory.