Potent anti-HIV Activity Research Articles

Exploring American Elderberry Compounds for Antioxidant, Antiviral, and Antibacterial Properties Through High-Throughput Screening Assays Combined with Untargeted Metabolomics.

American elderberry (Sambucus nigra subsp. canadensis) is a rapidly emerging new perennial crop for Missouri, recognized for its high level of bioactive compounds with significant health benefits, including antibacterial, antiviral, and antioxidant properties. A high-throughput screening assay combined with untargeted metabolomics analysis was utilized on American elderberry juice from 21 genotypes to explore and characterize these bioactive compounds. Our metabolomics study has identified 32 putative bioactive compounds in the American Elderberry juices. An array of high-throughput screening bioassays was conducted to evaluate 1) total antioxidant capacity, 2) activation of antioxidant response elements (ARE), 3) antiviral activity, and 4) antibacterial activity of the putatively identified compounds. Our results revealed that 14 of the 32 American elderberry compounds exhibited strong antioxidant activity. Four compounds (isorhamnetin 3-O-glucoside, kaempferol, quercetin, and naringenin) activated ARE activity and were found to be non-cytotoxic to cells. Notably, six of the 32 compounds demonstrated significant antiviral activity in an in vitro TZM-bl assay against two strains of HIV-1 virus, CXCR4-dependent NL4-3 virus and CCR5-dependent BaL virus. Luteolin showed the most potent anti-HIV activity against the NL4-3 virus (IC50 = 1.49 μM), followed by isorhamnetin (IC50 = 1.67 μM). The most potent anti-HIV compound against the BaL virus was myricetin (IC50 = 1.14 μM), followed by luteolin (IC50 = 4.38 μM). Additionally, six compounds were found to have antibacterial activity against gram-positive bacteria S. aureus, with cyanidin 3-O-rutinoside having the most potent antibacterial activity in vitro (IC50 = 2.9 μM), followed by cyanidin 3-O-glucoside (IC50 = 3.7 μM). These findings support and validate the potential health benefits of compounds found in American elderberry juices and highlight their potential for use in dietary supplements as well as innovative applications in health and medicine.

Daphnane diterpenoid orthoesters with an odd-numbered aliphatic side chain from Daphne pedunculata.

Daphnane diterpenoids were recognized for their extensive range of potent biological activities. In the present study, phytochemical investigation including LC-MS/MS analysis resulted in the identification of five daphnane diterpenoid orthoesters (1-5). Among the five daphnane diterpenoids, two previously unreported compounds, daphnepedunins I and J (2 and 4) were isolated from Daphne pedunculata. The structure of new compounds was elucidated with extensive physicochemical and spectroscopic analyses. Their structure was characterized by the presence of an unusual odd-numbered aliphatic chain connected to an orthoester. The isolated compounds were evaluated for their anti-HIV activity against HIV-1 infection of MT4 cells, and the results indicated that compound 1 showed the most potent anti-HIV activity with an IC50 value of 0.82nM.

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae.

Betulinic acid (BA) is a lupane-type triterpenoid with potent anticancer and anti-HIV activities. Its great potential in clinical applications necessitates the development of an efficient strategy for BA synthesis. This study attempted to achieve efficient BA biosynthesis in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, a de novo BA biosynthesis pathway in S. cerevisiae was constructed, which yielded a titer of 14.01 ± 0.21 mg/L. Then, by enhancing the BA synthesis pathway and dynamic inhibition of the competitive pathway, a greater proportion of the metabolic flow was directed toward BA synthesis, achieving a titer of 88.07 ± 5.83 mg/L. Next, acetyl-CoA and NADPH supply was enhanced, which increased the BA titer to 166.43 ± 1.83 mg/L. Finally, another BA synthesis pathway in the peroxisome was constructed. Dual regulation of the peroxisome and cytoplasmic metabolism increased the BA titer to 210.88 ± 4.76 mg/L. Following fed-batch fermentation process modification, the BA titer reached 682.29 ± 8.16 mg/L. Overall, this work offers a guide for building microbial cell factories that are capable of producing terpenoids with efficiency.

Late-Stage Derivatization of Oleanolic Acid-Based Anti-HIV-1 Compounds.

A 12-keto-type oleanolic acid derivative (4) has been identified as a potent anti-human immunodeficiency virus type-1 (HIV-1) compound that demonstrates synergistic effects with several types of HIV-1 neutralizing antibodies. In the present study, we used a common key synthetic intermediate to carry out the late-stage derivatization of an anti-HIV compound based on the chemical structure of a 12-keto-type oleanolic acid derivative. To execute this strategy, we designed a diketo-type oleanolic acid derivative (5) for chemoselective transformation, targeting the carboxy group and the hydroxyl group on the statine unit, as well as the 3-carbonyl group on the oleanolic acid unit, as orthogonal synthetic handles. We carried out four types of chemoselective transformations, leading to identification of the indole-type derivative (16) as a novel potent anti-HIV compound. In addition, further optimization of the β-hydroxyl group on the statine unit provided the R-4-isobutyl γ-amino acid-type derivative (6), which exhibited potent anti-HIV activity comparable to that of 4 but with reduced cytotoxicity.

Chemoenzymatic total synthesis of sorbicillactone A

The sorbicillinoid family is a large class of natural products known for their structural variety and strong, diverse biological activities. A special member of this family, sorbicillactone A, the first nitrogen-containing sorbicillinoid, exhibits potent anti-leukemic and anti-HIV activities and possesses a unique structure formed from sorbicillinol, alanine, and fumaric acid building blocks. To facilitate in-depth biological and structure-activity relationship studies of this promising natural product, we developed a chemoenzymatic approach that provides access to sorbicillactone A and several analogs with excellent yields under precise stereochemical control. The key steps of the highly convergent, stereoselective, and short route are the enantioselective oxidative dearomatization of sorbillin to sorbicillinol catalyzed by the enzyme SorbC and the subsequent Michael addition of a fumarylazlactone building block. Additionally, our synthetic findings and bioinformatic analysis suggest that sorbicillactone A is biosynthetically formed analogously.

Total Synthesis and Anti-HIV Activity Evaluation of Desmosdumotin D and Analogues

The natural product Desmosdumotin D (hereafter referred to as Des-D), isolated from the plant Desmos dumosus, showed potent anti-HIV activity. However, the subsequent pharmacological activity and clinical studies are limited due to the low content of Des-D in the plant. Therefore, the total synthesis path of Des-D was optimized in this paper, and the total yield was increased from 4.4% to 11.9%. Additionally, twelve analogues were obtained following the synthesis route of Des-D. The anti-HIV activity evaluation results in vitro showed that Des-D had the highest activity, with an IC50 value of 13.6 μM, and compounds 17 and 11 had the lowest anti-HIV activity, with IC50 values of 101.3 μM and 161.0 μM, respectively. Through the molecular docking of compounds Des-D and 17 with HIV-IN, the results show that phenolic hydroxyl groups and two benzene rings interact with HIV-IN and are possible pharmacodynamic groups.

Broad-spectrum anti-HIV activity and high drug resistance barrier of lipopeptide HIV fusion inhibitor LP-19.

Lipopeptide-19, a HIV fusion inhibitor (LP-19), has showed potent anti-HIV activity. However, there is still limited information of the antiviral activity against different subtype clinical isolates and the drug resistance barrier of LP-19. Therefore, 47 HIV clinical isolates were selected for this study. The viral features were identified, in which 43 strains are CCR5 tropisms, and 4 strains are CCR5/CXCR4 tropisms, and there are 6 subtype B', 15 CRF01_AE, 14 CRF07_BC, 2 CRF08_BC and 10 URF strains. These 47 viruses were used to detected and analyze the inhibitory activities of LP-19. The results showed that the average 50% inhibitory concentration (IC50) and 90% inhibitory concentration (IC90) of LP-19 were 0.50 nM and 1.88 nM, respectively. The average IC50 of LP-19 to B', CRF01_AE, CRF07_BC, CRF08_BC, and URF strains was 0.76 nM, 0.29 nM, 0.38 nM, 0.85 nM, and 0.44 nM, respectively. C34 and Enfuvirtide (T-20), two fusion inhibitors, were compared on the corresponding strains simultaneously. The antiviral activity of LP-19 was 16.7-fold and 86-fold higher than that of C34 and T-20. The antiviral activity of LP-19, C34, and T-20 were further detected and showed IC50 was 0.15 nM, 1.02 nM, and 66.19 nM, respectively. IC50 of LP-19 was about 7-fold and 441-fold higher compared to C34 and T-20 against HIV-1 NL4-3 strains. NL4-3 strains were exposed to increasing concentrations of LP-19 and C34 in MT-2 cell culture. The culture virus was sequenced and analyzed. The results showed that A243V mutation site identified at weeks 28, 32, 38, and 39 of the cell culture in the gp41 CP (cytoplasmic domain) region. NL4-3/A243V viruses containing A243V mutation were constructed. Comparing the antiviral activities of LP-19 against HIV NL4-3 to HIV strains (only 1.3-fold), HIV did not show drug resistance when LP-19 reached 512-fold of the initial concentration under the drug pressure for 39 weeks. This study suggests that LP-19 has broad-spectrum anti-HIV activity, and high drug resistance barrier.

Stelleratenoids A−F, macrocyclic daphnane orthoesters with anti-HIV activity from the roots of Stellera chamaejasme L.

Six undescribed macrocyclic daphnane orthoesters, stelleratenoids A−F (1−6), were isolated from the roots of Stellera chamaejasme L. Their structures were elucidated by extensive spectroscopic analyses, including HRESIMS and NMR spectra. Compound 1 features an unusual terminal double bond at C-2/C-19 in the 1α-alkyldaphnane lactone skeleton. Compounds 2−4 are unique in the presence of different long chain fatty acyl groups. Compounds 5 and 6 are unique examples of modified macrocyclic daphnane diterpenoids. All the isolates were evaluated for anti-HIV activity in MT-2 cells. Among them, compounds 1, 5 and 6 exhibited highly potent anti-HIV activity with EC50 values of 66.70, 10.62 and 55.10 nM, respectively, possessing high potential to develop new anti-HIV drugs.

Synthesis and Cytotoxic Mechanism of Sulfated Alkyl Oligosaccharides Having Potent Anti-HIV Activity

Synthesis and Cytotoxic Mechanism of Sulfated Alkyl Oligosaccharides Having Potent Anti-HIV Activity

Anti-HIV Macrocyclic Daphnane Orthoesters with an Unusual Macrocyclic Ring from Edgeworthia chrysantha.

Edgeworthianins A-E (1-5) were isolated from Edgeworthia chrysantha as a class of macrocyclic daphnane orthoesters with an unusual macrocyclic ring formed from a C14 aliphatic chain. Their structures were elucidated by extensive physicochemical and spectroscopic analyses. Compounds 2, 4, and 5 exhibited potent anti-HIV activity against HIV-1 infection of MT4 cells with EC50 values of 29.3, 8.4, and 2.9 nM, respectively. These compounds broaden the findings of the structure-activity relationship of macrocyclic daphnane orthoesters for further anti-HIV drug development.

Ursane and 24-Noroleanane-Type Triterpenoids with Anti-HIV Activity from the Twigs and Leaves of Antirhea chinensis.

Investigations on the twigs and leaves of Antirhea chinensis have led to the discovery of two undescribed pentacyclic triterpenoids (1 and 2) and nine known analogs. Compounds 1 and 2, each assigned as the ursane and 24-noroleanane-type triterpenoids, featuring similar oxidation pattern of 3β,6β,19α-trihydroxy-28-carboxyl. Their structures were elucidated via comprehensive analyses of spectroscopic data. Compound 1 displayed potent anti-HIV activity (EC50 =1.24 μM) and high selectivity index (SI >32.3).

From design to biological mechanism evaluation of phenylalanine-bearing HIV-1 capsid inhibitors targeting a vital assembly interface

HIV-1 capsid protein (CA) has emerged as a promising target for antiviral treatment considering its structural and regulatory roles in HIV-1 replication. Here, we disclose the design, synthesis, biological assessment, and mechanism investigation of a novel series of phenylalanine derivatives gained by further structural modification of PF74. The newly synthesized compounds demonstrated potent anti-HIV activity, represented by 7n displayed anti-HIV-1 activity 6.25-fold better than PF74, and 7h showed anti-HIV-2 activity with nearly 139 times improved efficacy over PF74. Surface plasmon resonance (SPR) studies of representative compounds proved that HIV-1 CA was the binding target. Competitive SPR studies using CPSF6 and NUP153 peptides identified that 7n binds to a vital CA assembly interface between the N-terminal and C-terminal domain (NTD-CTD interface). Action stage determination assay revealed that the newly synthesized compounds were antiviral with a dual-stage inhibitory profile. Molecular dynamics (MD) simulations offered the crucial foundation for the hopeful antiviral potency of 7n. Besides, 7m and 7n modestly increased metabolic stabilities in human liver microsome (HLM) and human plasma compared to PF74. Overall, these studies offer valuable insights and can regard as the beginning for succedent medicinal chemistry endeavors to discover promising HIV capsid inhibitors with improved efficacy and better drug-like characteristics.

Constituents of Croton megalocarpus with Potential Anti-HIV Activity.

Reported herein is an anti-HIV monochlorinated compound, 1β-acetoxy-3β-chloro-5α,6α-dihydroxycrotocascarin L (1), of the rare crotofolane diterpenoid class. Compound 1, a suspected artifact of extraction, along with the previously undescribed 11β-acetoxycrotocascarin L (2) and a known compound, crotocascarin K (3), were isolated from the bark of Croton megalocarpus, a Kenyan oil-producing seed crop. Compounds 1 and 3 inhibited HIV-1 replication with IC50 values of 28 and 5.5 nM, respectively. Furthermore, both compounds lacked cytotoxicity toward MT-4 cells and FM-55-M1 cells at concentrations of up to 50 μM. Compounds 1 and 3 were both found to inhibit HIV-1 protease.

In vitro anti-HIV and cytotoxic effects of pure compounds isolated from Croton macrostachyus Hochst. Ex Delile

Croton macrostachyus is an important plant in traditional African medicine, widely utilized to treat a variety of diseases. In Kenya, HIV-infected patients use leaf and root decoctions of the plant as a cure for cough, back pain, bleeding, skin diseases, warts, pneumonia, and wounds. This study aimed to evaluate the anti-HIV activities and cytotoxic effects of extracts and chemical constituents isolated from C. macrostachyus. In our previous study we demonstrated that the hexane, CH2Cl2, ethyl acetate and methanol soluble fractions of a 1:1 v/v/ CH2Cl2/MeOH crude extracts of the leaves and stem bark of C. macrostachyus exhibited potent anti-HIV activities against HIV-1 with IC50 values ranging from 0.02–8.1 μg/mL and cytotoxicity effects against MT-4 cells ranging from IC50 = 0.58–174 μg/mL. Hence, hexane soluble extract of 1:1 v/v/ CH2Cl2/MeOH crude extract of the leaves of C. macrostachyus, that was more potent against HIV-1 at IC50 = 0.02 μg/mL was subjected to column chromatography leading to the isolation of 2-methoxy benzyl benzoate (1), lupenone (2), lupeol acetate (3), betulin (4), lupeol (5), sitosterol (6) and stigmasterol (7). Lupenone (2), lupeol acetate (3) and betulin (4) exhibited anti-HIV-1 inhibition at IC50 = 4.7 nM, 4.3 and 4.5 μg/mL respectively. The results obtained from this study support the potential of C. macrostachyus, as a source of anti-HIV constituents.

Anti-HIV Tigliane-Type Diterpenoids from the Aerial Parts of Wikstroemia lichiangensis.

Tigliane-type diterpenoids have attracted much attention in drug discovery since they have been reported to exhibit remarkable biological effects, such as tumor-promoting, antineoplastic, and anti-HIV activities. In continuing our efforts to discover novel biologically important diterpenoids from Wikstroemia species, Wikstroemia lichiangensis was investigated phytochemically for the first time. As a result, four new (1-4) and one known (5) tigliane-type diterpenoid were isolated, and their structures were elucidated by spectroscopic data analysis. Tiglianes (1-5) showed potent anti-HIV activity against HIV-1 infection of MT4 lymphocytes with IC50 values of 1.1-65.4 nM.

C3-Symmetric Aromatic Core of Griffithsin Is Essential for Potent Anti-HIV Activity.

Lectins, carbohydrate-binding proteins of nonimmune origin, bind to carbohydrates and glycan shields present on the surfaces of cells and viral spike proteins. Lectins thus hold great promise as therapeutic and diagnostic proteins, exemplified by their potent antiviral activities and the desire to engineer synthetic carbohydrate receptors based on lectin recognition principles. Here, we describe a new carbohydrate-binding architectural motif─namely, a C3-symmetric tyrosine-based aromatic core, present in the therapeutic lectin griffithsin (GRFT). By using structure-based amino acid substitutions, X-ray crystallography, molecular dynamics (MD) simulations, and HIV-1 neutralization assays, we show that this core is critical for potent (pM) antiviral activity and nanomolar binding to the glycan shield largely consisting of high mannose glycans. Crystal structures and MD simulations show that CH-π interactions stabilize the aromatic cluster to maintain the three pseudo-symmetric carbohydrate-binding sites, nonaromatic amino acid substitutions (Tyr to Ala) abrogate antiviral activity, and increasing the aromatic CH-π edge-to-centroid interface via a Tyr to Trp substitution yields a GRFT variant with improved potency and increased residence time of Man-9 observed in MD simulations. NMR titrations of a Tyr-to-Ala variant indicate that disruption of the aromatic prevents the intermolecular crosslinking between two equivalents of Man-9 and one carbohydrate-binding face observed in wild-type GRFT and known to be critical for picomolar potency of this lectin. This C3-symmetric aromatic core defines a new recognition motif for the design of carbohydrate receptors and suggests principles for engineering known lectins to have increased affinity and stability.

Exploratory studies on soluble small molecule CD4 mimics as HIV entry inhibitors

Several small molecule CD4 mimics, which inhibit the interaction of gp120 with CD4, have been developed. Original CD4 mimics such as NBD-556, which has an aromatic ring, an oxalamide linker and a piperidine moiety, possess significant anti-HIV activity but with their hydrophobic aromatic ring-containing structures are poorly soluble in water. We have developed derivatives with a halopyridinyl group in place of the phenyl group, such as KKN-134, and found them to have excellent aqueous solubility. Other leads that were examined are YIR-821, a compound with a cyclohexane group in a spiro attachment to a piperidine ring and a guanidino group on the piperidine nitrogen atom, and its PEGylated derivative, TKB-002. YIR-821 and TKB-002 retain potent anti-HIV activity. Here, new CD4 mimics, in which the phenyl group was replaced by a halopyridinyl group with the halogen atoms in different positions, their derivatives without a cyclohexane group on the piperidine ring and their hybrid molecules with PEG units were designed and synthesized. Some of these compounds show significantly higher aqueous solubility with maintenance of certain levels of anti-HIV activity. The present data should be useful in the future design of CD4 mimic molecules.

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy

ABSTRACT Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell–cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

Clausanisumine, a Prenylated Bicarbazole Alkaloid from the Fruits of Clausena anisum-olens and Its Potential Anti-HIV Activity.

A unique prenylated bicarbazole alkaloid, clausanisumine (1), and two biogenetically related known monomer carbazole alkaloids, mukonal (2) and 3-methylcarbazole (3), were isolated from the fruits of Clausena anisum-olens. Clausanisumine (1) was an uncommon prenylated bicarbazole alkaloid, possessing an unprecedented carbon skeleton, which was composed of a simple carbazole alkaloid and a prenylated carbazole alkaloid. The chemical structure of 1 was established by a combination of comprehensive spectral methods. A plausible biosynthetic pathway of 1 was also proposed. Additionally, the potential anti-HIV activities of all isolates 1-3 in vitro were evaluated. Compound 1 exhibited remarkable anti-HIV-1 reverse transcriptase effects showing an EC50 value of 18.58 nM. The discovery of the prenylated bicarbazole alkaloid from C. anisum-olens with notable anti-HIV activity would be meaningful to discovering and developing new anti-HIV drugs.

ω-Functionalized Lipid Prodrugs of HIV NtRTI Tenofovir with Enhanced Pharmacokinetic Properties.

Tenofovir (TFV) is the cornerstone nucleotide reverse transcriptase inhibitor (NtRTI) in many combination antiretroviral therapies prescribed to patients living with HIV/AIDS. Due to poor cell permeability and oral bioavailability, TFV is administered as one of two FDA-approved prodrugs, both of which metabolize prematurely in the liver and/or plasma. This premature prodrug processing depletes significant fractions of each oral dose and causes toxicity in kidney, bone, and liver with chronic administration. Although TFV exalidex (TXL), a phospholipid-derived prodrug of TFV, was designed to address this issue, clinical pharmacokinetic studies indicated substantial hepatic extraction, redirecting clinical development of TXL toward HBV. To circumvent this metabolic liability, we synthesized and evaluated ω-functionalized TXL analogues with dramatically improved hepatic stability. This effort led to the identification of compounds 21 and 23, which exhibited substantially longer t1/2 values than TXL in human liver microsomes, potent anti-HIV activity in vitro, and enhanced pharmacokinetic properties in vivo.