Towards the De Novo Design of HIV-1 Protease Inhibitors Based on Natural Products.

Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) continues to be a public health problem. In 2020, 680,000 people died from HIV-related causes, and 1.5 million people were infected. Antiretrovirals are a way to control HIV infection but not to cure AIDS. As such, effective treatment must be developed to control AIDS. Developing a drug is not an easy task, and there is an enormous amount of work and economic resources invested. For this reason, it is highly convenient to employ computer-aided drug design methods, which can help generate and identify novel molecules. Using the de novo design, novel molecules can be developed using fragments as building blocks. In this work, we develop a virtual focused compound library of HIV-1 viral protease inhibitors from natural product fragments. Natural products are characterized by a large diversity of functional groups, many sp3 atoms, and chiral centers. Pseudo-natural products are a combination of natural products fragments that keep the desired structural characteristics from different natural products. An interactive version of chemical space visualization of virtual compounds focused on HIV-1 viral protease inhibitors from natural product fragments is freely available in the supplementary material.

Accessing HIV-1 Protease Inhibitors through Visible-Light-Mediated Sequential Photocatalytic Decarboxylative Radical Conjugate Addition-Elimination-Oxa-Michael Reactions.

A photocatalytic decarboxylative radical conjugate addition-elimination-oxa-Michael reaction of hydroxyalkylated carboxylic acids with cyclopentenones is developed to construct diverse cyclopentanonyl-fused functionalized 5-7 membered cyclic ethers. The stereoselective synthetic strategy is amenable to substructural variation, establishing a direct total synthetic route to two diastereomers of C3-amino cyclopentyltetrahydrofuranyl-derived potent HIV-1 protease inhibitors with low nanomolar IC50 values.

Novel pyridinium-type fullerene derivatives as multitargeting inhibitors of HIV-1 reverse transcriptase, HIV-1 protease, and HCV NS5B polymerase.

In the present study, we newly synthesized four types of novel fullerene derivatives: pyridinium/ethyl ester-type derivatives 3b-3l, pyridinium/carboxylic acid-type derivatives 4a, 4e, 4f, pyridinium/amide-type derivative 5a, and pyridinium/2-morpholinone-type derivative 6a. Among the assessed compounds, cis-3c, cis-3d, trans-3e, trans-3h, cis-3l, cis-4e, cis-4f, trans-4f, and cis-5a were found to inhibit HIV-1 reverse transcriptase (HIV-RT), HIV-1 protease (HIV-PR), and HCV NS5B polymerase (HCV NS5B), with IC50 values observed in the micromolar range. Cellular uptake of pyridinium/ethyl ester-type derivatives was higher than that of corresponding pyridinium/carboxylic acid-type derivatives and pyridinium/amide-type derivatives. This result might indicate that pyridinium/ethyl ester-type derivatives are expected to be lead compounds for multitargeting drugs to treat HIV/HCV coinfection.

Design and biological evaluation of cinnamic and phenylpropionic amide derivatives as novel dual inhibitors of HIV-1 protease and reverse transcriptase.

Upon the basis of both possible ligand-binding site interactions and the uniformity of key residues in active sites, a novel class of HIV-1 PR/RT dual inhibitors was designed and evaluated. Cinnamic acids or phenylpropionic acids with more flexible chain and smaller steric hindrance were introduced into the inhibitors, giving rise to significant improvement in HIV-1 RT inhibitory activity by one or two orders of magnitude, with comparable or even improved potency against PR at the same time, compared with coumarin anologues in our previous studies. Among these inhibitors, 38d displayed a 19-fold improvement in anti-PR activity with IC50 value of 0.081 nM compared to the control DRV. In addition, inhibitor 38c exhibited an excellent anti-RT IC50 value of 0.43 µM, only a 4.7-fold less potent activity than the control EFV. More significantly, the disparate ratio between HIV-1 PR and RT inhibition became more reasonable with ratio of 1: 10.4, just as 37b. Furthermore, the assays on HIV-1 late stage and early stage supported the rationality of designing dual inhibitors. The SAR data as well as molecular modeling studies provided new insight for further optimization of more potent HIV-1 PR/RT dual inhibitors.

Design and evaluation of novel piperidine HIV-1 protease inhibitors with potency against DRV-resistant variants.

A novel class of HIV-1 protease inhibitors with flexible piperidine as the P2 ligand was designed with the aim of improving extensive interactions with the active subsites. Many inhibitors exhibited good to excellent inhibitory effect on enzymatic activity and viral infectivity. In particular, inhibitor 3a with (R)-piperidine-3-carboxamide as the P2 ligand and 4-methoxybenzenesulfonamide as the P2' ligand showed an enzyme Ki value of 29 pM and antiviral IC50 value of 0.13 nM, more than six-fold enhancement of activity compared to DRV. Furthermore, there was no significant change in potency against DRV-resistant mutations and HIV-1NL4-3 variant for 3a. Besides, inhibitor 3a exhibited potent antiviral activity against subtype C variants with low nanomole EC50 values. In addition, the molecular modeling revealed important hydrogen bonds and other favorable van der Waals interactions with the backbone atoms of the protease and provided insight for designing and optimizing more potent HIV-1 protease inhibitors.

Fullerene derivatives as dual inhibitors of HIV-1 reverse transcriptase and protease.

In the present study, we newly synthesized three types of novel fullerene derivatives: pyridinium-type derivatives trans-3a and 4a-5b, piperidinium-type derivative 9, and proline-type derivatives 10a-12. Among the assessed compounds, 5a, 10e, 10f, 10i, 11a-d, and 12 were found to inhibit both HIV reverse transcriptase and HIV protease (HIV-PR), with IC50 values in the low micromolar range being observed. Regarding HIV-PR inhibition activity, proline-type derivatives 11a-11d and 12, bearing an alkyl chain between the hydroxylmethylcarbonyl (HMC) moiety and pyrrolidine ring, were more potent than other derivatives. This result might indicate that connecting HMC moieties with proline-type fullerene derivatives through properly sized alkyl chain leads to improved HIV-PR inhibitory activity.

Piperidine scaffold as the novel P2-ligands in cyclopropyl-containing HIV-1 protease inhibitors: Structure-based design, synthesis, biological evaluation and docking study.

A series of potent HIV-1 protease inhibitors, containing diverse piperidine analogues as the P2-ligands, 4-substituted phenylsulfonamides as the P2'-ligands and a hydrophobic cyclopropyl group as the P1'-ligand, were designed, synthesized and evaluated in this work. Among these twenty-four target compounds, many of them exhibited excellent activity against HIV-1 protease with half maximal inhibitory concentration (IC50) values below 20 nM. Particularly, compound 22a containing a (R)-piperidine-3-carboxamide as the P2-ligand and a 4-methoxylphenylsulfonamide as the P2'-ligand exhibited the most effective inhibitory activity with an IC50 value of 3.61 nM. More importantly, 22a exhibited activity with inhibition of 42% and 26% against wild-type and Darunavir (DRV)-resistant HIV-1 variants, respectively. Additionally, the molecular docking of 22a with HIV-1 protease provided insight into the ligand-binding properties, which was of great value for further study.

Development of Darunavir proliposome powder for oral delivery by using Box-Bhenken design.

The aim of this study is to develop Darunavir (DRV) proliposome powder for oral delivery. Darunavir-loaded oral proliposome powder (OPP) was prepared by a solvent evaporation technique with varying independent variables at three different levels. Based on different levels, proliposome powder formulation was optimized by using Box-Behnken design. The formulations were analyzed for its size distribution, entrapment efficiency, and surface morphology. Optimized proliposome batch A was evaluated for physical parameter, morphological parameters, entrapment efficiency, followed by in vitro, ex vivo, and in vivo studies. Oral proliposome powder showed good micromeritic properties with angle of repose was less than 30°, Carr's index and Hausner's ratio were also less than 21 and 1.25, respectively. The mean size of the vesicles was in the range of 180-290 nm. The assay and entrapment efficiency of pro-liposome powder formulations were 79.00 ± 0.2 and 93.46 ± 0.2%, respectively. In vitro release of DRV proliposome powder was 78.17 ± 0.1% after 24 h which shows good release from the vesicle of proliposome. Ex vivo permeation study shows 58.11% enhancement which shows good permeation. The optimize batch A of proliposome powder indicated 50% enhancement in the relative bioavailability as compared to the DRV suspension. The results showed that proliposome powder containing DRV can efficiently deliver in to the blood stream. This drug delivery system has been designed as a novel platform for potential oral delivery of drugs having poor water solubility and high first-pass metabolism.

Structure-Based Design of Highly Potent HIV-1 Protease Inhibitors Containing New Tricyclic Ring P2-Ligands: Design, Synthesis, Biological, and X-ray Structural Studies.

We describe here design, synthesis, and biological evaluation of a series of highly potent HIV-1 protease inhibitors containing stereochemically defined and unprecedented tricyclic furanofuran derivatives as P2 ligands in combination with a variety of sulfonamide derivatives as P2' ligands. These inhibitors were designed to enhance the ligand-backbone binding and van der Waals interactions in the protease active site. A number of inhibitors containing the new P2 ligand, an aminobenzothiazole as the P2' ligand and a difluorophenylmethyl as the P1 ligand, displayed very potent enzyme inhibitory potency and also showed excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The tricyclic P2 ligand has been synthesized efficiently in an optically active form using enzymatic desymmetrization of meso-1,2-(dihydroxymethyl)cyclohex-4-ene as the key step. We determined high-resolution X-ray structures of inhibitor-bound HIV-1 protease. These structures revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insights into the binding properties of these new inhibitors.

Synthesis and evaluation of potent human immunodeficiency virus 1 protease inhibitors with epimeric isopropanol as novel P1' ligands.

Aim: HIV-1 protease inhibitors regimens suffered from a number of drawbacks, among which, the most egregious issue was the growing emergence of drug-resistant strains. Materials & methods: The design strategy of maximizing the protease active site interactions with the inhibitor, especially promoting extensive hydrogen bonding with the protein backbone atoms, might be in favor of combating drug resistance. A series of HIV-1 protease inhibitors that incorporated enantiomeric isopropanols as the P1' ligands in combination with phenols as the P2 ligands were reported herein. Results: A number of inhibitors displayed potent protease enzyme inhibition activity. In particular, inhibitor 14c showed comparable potency as darunavir with IC50 value of 1.91 nM and activity against darunavir-resistant HIV-1 variants. Conclusion: The new kind of HIV-1 protease inhibitors deserves further study.

Rh(iii)-Catalyzed diastereoselective transfer hydrogenation: an efficient entry to key intermediates of HIV protease inhibitors.

A highly efficient diastereoselective transfer hydrogenation of α-aminoalkyl α'-chloromethyl ketones catalyzed by a tethered rhodium complex was developed and successfully utilized in the synthesis of the key intermediates of HIV protease inhibitors. With the current Rh(iii) catalyst system, a series of chiral 3-amino-1-chloro-2-hydroxy-4-phenylbutanes were produced in excellent yields and diastereoselectivities (up to 99% yield, up to 99 : 1 dr). Both diastereomers of the desired products could be efficiently accessed by using the two enantiomers of the Rh(iii) catalyst.

Design, synthesis and biological evaluation of HIV-1 protease inhibitors with morpholine derivatives as P2 ligands in combination with cyclopropyl as P1' ligand.

A series of novel HIV-1 protease inhibitors has been designed and synthesized, which contained morpholine derivatives as the P2 ligands and hydrophobic cyclopropyl as the P1' ligand at the meantime in this study, with the aim of improving the interactions between the active sites of HIV-1 protease and the inhibitors. Twenty-eight compounds were synthesized and assessed, among which inhibitors m18 and m1 exhibited excellent inhibitory effect on the activity of HIV-1 protease with IC50 value of 47 nM and 53 nM, respectively. The molecular modeling of m1 revealed possible hydrogen bondings or van der Waals between the inhibitor and the protease, worthy of in-depth study.

Aspartic peptidase of Phialophora verrucosa as target of HIV peptidase inhibitors: blockage of its enzymatic activity and interference with fungal growth and macrophage interaction.

Phialophora verrucosa causes several fungal human diseases, mainly chromoblastomycosis, which is extremely difficult to treat. Several studies have shown that human immunodeficiency virus peptidase inhibitors (HIV-PIs) are attractive candidates for antifungal therapies. This work focused on studying the action of HIV-PIs on peptidase activity secreted by P. verrucosa and their effects on fungal proliferation and macrophage interaction. We detected a peptidase activity from P. verrucosa able to cleave albumin, sensitive to pepstatin A and HIV-PIs, especially lopinavir, ritonavir and amprenavir, showing for the first time that this fungus secretes aspartic-type peptidase. Furthermore, lopinavir, ritonavir and nelfinavir reduced the fungal growth, causing remarkable ultrastructural alterations. Lopinavir and ritonavir also affected the conidia-macrophage adhesion and macrophage killing. Interestingly, P. verrucosa had its growth inhibited by ritonavir combined with either itraconazole or ketoconazole. Collectively, our results support the antifungal action of HIV-PIs and their relevance as a possible alternative therapy for fungal infections.

Molecular Docking, Synthesis and anti-HIV-1 Protease Activity of Novel Chalcones.

BACKGROUND: Since the beginning of the HIV/AIDS epidemic, 75 million people have been infected with the HIV and about 32 million people have died of AIDS. Investigation of the molecular mechanisms critical to the HIV replication cycle led to the identification of potential drug targets for AIDS therapy. One of the most important discoveries is HIV-1 protease, an enzyme that plays an essential role in the replication cycle of HIV. OBJECTIVE: The aim of the present study is to synthesize and investigate anti-HIV-1 protease activity of some chalcone derivatives with the hope of discovering new lead structure devoid drug resistance. METHODS: 20 structurally similar chalcone derivatives were synthesized and their physico-chemical characterization was performed. Binding of chalcones to HIV-1 protease was investigated by fluorimetric assay. Molecular docking studies were conducted to understand the interactions. RESULTS: The obtained results revealed that all compounds showed anti-HIV-1 protease activity. Compound C1 showed the highest inhibitory activity with an IC50 value of 0.001 µM, which is comparable with commercial product Darunavir. CONCLUSION: It is difficult to provide general principles of inhibitor design. Structural properties of the compounds are not the only consideration; ease of chemical synthesis, low molecular weight, bioavailability, and stability are also of crucial importance. Compared to commercial products the main advantage of compound C1 is the ease of chemical synthesis and low molecular weight. Furthermore, compound C1 has a structure that is different to peptidomimetics, which could contribute to its stability and bioavailability.

Rational design and Structure-Activity relationship of coumarin derivatives effective on HIV-1 protease and partially on HIV-1 reverse transcriptase.

Since dual inhibitors may yield lower toxicity and reduce the likelihood of drug resistance, as well as inhibitors of HIV-1 PR and RT constitute the core of chemotherapy for AIDS treatment, we herein designed and synthesized new coumarin derivatives characterized by various linkers that exhibited excellent potency against PR and a weak inhibition of RT. Among which, compounds 6f and 7c inhibited PR with IC50 values of 15.5 and 62.1 nM, respectively, and weakly affected also RT with IC50 values of 241.8 and 188.7 µM, respectively, showing the possibility in the future of developing dual HIV-1 PR/RT inhibitors. Creative stimulation for further research of more potent dual HIV-1 inhibitors was got according to the molecular docking studies.

Potent HIV-1 Protease Inhibitors Containing Carboxylic and Boronic Acids: Effect on Enzyme Inhibition and Antiviral Activity and Protein-Ligand X-ray Structural Studies.

We report the synthesis and biological evaluation of phenylcarboxylic acid and phenylboronic acid containing HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. Inhibitors bearing bis-THF ligand as P2 ligand and phenylcarboxylic acids and carboxamide as the P2' ligands, showed very potent HIV-1 protease inhibitory activity. However, carboxylic acid containing inhibitors showed very poor antiviral activity relative to carboxamide-derived inhibitors which showed good antiviral IC50 value. Boronic acid derived inhibitor with bis-THF as the P2 ligand showed very potent enzyme inhibitory activity, but it showed lower antiviral activity than darunavir in the same assay. Boronic acid containing inhibitor with a P2-Crn-THF ligand also showed potent enzyme Ki but significantly decreased antiviral activity. We have evaluated antiviral activity against a panel of highly drug-resistant HIV-1 variants. One of the inhibitors maintained good antiviral activity against HIVDRVRP20 and HIVDRVRP30 viruses. We have determined high resolution X-ray structures of two synthetic inhibitors bound to HIV-1 protease and obtained molecular insight into the ligand-binding site interactions.

HIV-1 Protease Inhibitors Incorporating Stereochemically Defined P2' Ligands To Optimize Hydrogen Bonding in the Substrate Envelope.

A structure-guided design strategy was used to improve the resistance profile of HIV-1 protease inhibitors by optimizing hydrogen bonding and van der Waals interactions with the protease while staying within the substrate envelope. Stereoisomers of 4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties were explored as P2' ligands providing pairs of diastereoisomers epimeric at P2', which exhibited distinct potency profiles depending on the configuration of the hydroxyl group and size of the P1' group. While compounds with the 4-(1-hydroxyethyl)benzene P2' moiety maintained excellent antiviral potency against a panel of multidrug-resistant HIV-1 strains, analogues with the polar 4-(1,2-dihydroxyethyl)benzene moiety were less potent, and only the (R)-epimer incorporating a larger 2-ethylbutyl P1' group showed improved potency. Crystal structures of protease-inhibitor complexes revealed strong hydrogen bonding interactions of both (R)- and (S)-stereoisomers of the hydroxyethyl group with Asp30'. Notably, the (R)-dihydroxyethyl group was involved in a unique pattern of direct hydrogen bonding interactions with the backbone amides of Asp29' and Asp30'. The SAR data and analysis of crystal structures provide insights for optimizing these promising HIV-1 protease inhibitors.

Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation.

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)-hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)-aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory Ki value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.

A Photochemical Route to Optically Active Hexahydro-4H-furopyranol, a High-Affinity P2 Ligand for HIV-1 Protease Inhibitors.

We describe here the syntheses of optically pure (3aS,4S,7aR)-hexahydro-4H-furo[2,3-b]pyran-4-ol and (3aR,4R,7aS)-hexahydro-4H-furo[2,3-b]pyran-4-ol. These stereochemically defined heterocycles are important high-affinity P2 ligands for a variety of highly potent HIV-1 protease inhibitors. The key steps involve an efficient Paternò-Büchi [2 + 2] photocycloaddition, catalytic hydrogenation, acid-catalyzed cyclization to form the racemic ligand alcohol, and an enzymatic resolution with immobilized Amano Lipase PS-30. Optically active ligands (-)-6 and (+)-6 were obtained with high enantiomeric purity. Enantiomer (-)-6 has been converted to potent HIV-1 protease inhibitor 3.

Design, Synthesis, and Pharmacokinetic Evaluation of Phosphate and Amino Acid Ester Prodrugs for Improving the Oral Bioavailability of the HIV-1 Protease Inhibitor Atazanavir.

Phosphate and amino acid prodrugs of the HIV-1 protease inhibitor (PI) atazanavir (1) were prepared and evaluated to address solubility and absorption limitations. While the phosphate prodrug failed to release 1 in rats, the introduction of a methylene spacer facilitated prodrug activation, but parent exposure was lower than that following direct administration of 1. Val amino acid and Val-Val dipeptides imparted low plasma exposure of the parent, although the exposure of the prodrugs was high, reflecting good absorption. Screening of additional amino acids resulted in the identification of an l-Phe ester that offered an improved exposure of 1 and reduced levels of the circulating prodrug. Further molecular editing focusing on the linker design culminated in the discovery of the self-immolative l-Phe-Sar dipeptide derivative 74 that gave four-fold improved AUC and eight-fold higher Ctrough values of 1 compared with oral administration of the drug itself, demonstrating a successful prodrug approach to the oral delivery of 1.