This mini review covers the drug discovery aspect of both proteasome activators and inhibitors. The proteasome is involved in many essential cellular functions, such as regulation of cell cycle, cell differentiation, signal transduction pathways, antigen processing for appropriate immune responses, stress signaling, inflammatory responses, and apoptosis. Due to the importance of the proteasome in cellular functions, inhibition or activation of the proteasome could become a useful therapeutic strategy for a variety of diseases. Many proteasome inhibitors have been identified and can be classified into two groups according to their source: chemically synthesized small molecules and compounds derived from natural products. A successful case of developing a proteasome inhibitor as a clinically useful drug is that the peptide boronate, PS341 (Bortezomib), was approved for the treatment of multiple myeloma. In contrast to proteasome inhibitors, small molecules that can activate or enhance proteasome activity are rare and are not well studied. The fact that over-expression of the cellular proteasome activator PA28 exhibited beneficial effects on the Huntington’s disease neuronal model cells raised the prospect that small molecule proteasome activators could become useful therapeutics. The beneficial effect of oleuropein, a small molecule proteasome activator, on senescence of human fibroblasts also suggested that proteasome activators might have the potential to be developed into anti-aging agents.
Proteasome; activator; inhibitor; natural products; betulinic acids; peptides; anti-cancer; bortezomib
HIV-1 envelope glycoproteins are the key viral proteins that mediate HIV-1 entry and cell–cell fusion. In contrast to HIV-1 entry, the mechanism of HIV-1 Env-mediated cell–cell fusion is relatively unclear. This study demonstrated that dynasore, a dynamin inhibitor, suppressed HIV-1 Env-mediated cell–cell fusion. Dynasore sensitivity of HIV-1 Env-mediated cell–cell fusion varied depending on the viral strains. Results from testing a panel of gp41 cytoplasmic tail truncation mutants suggested that the gp41 cytoplasmic tail might play a role in dynasore sensitivity. HIV-1 Env-mediated cell–cell fusion could also be suppressed by a dynamin dominant-negative mutant DNM2(K44A). In summary, these results suggested that dynamin 2 might play a role in HIV-1 Env-mediated cell–cell fusion.
A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of potency on proteasome inhibition. Among them, the phenylene diacetic acid hemiester derivative (13) displayed the most potent proteasome inhibition with IC50 = 1.9 μM. Enzyme kinetic analysis indicates that these lithocholic acid derivatives are non-competitive inhibitors of the proteasome.
Lithocholic acid; proteasome; proteasome inhibitor
This study discovered that glycyrrhetinic acid inhibited the human 20S proteasome at 22.3 µM. Esterification of the C-3 hydroxyl group on glycyrrhetinic acid with various carboxylic acid reagents yielded a series of analogs with marked improved potency. Among the derivatives, glycyrrhetinic acid 3-O-isophthalate (17) was the most potent compound with IC50 of 0.22 µM, which was approximately 100-fold more potent than glycyrrhetinic acid.
Glycyrrhetinic acid; proteasome inhibitor; triterpene
This study discovered that betulinic acid (BA) is a potent proteasome activator that preferentially activates the chymotrypsin-like activity of proteasomes. Chemical modifications can transform BA into proteasome inhibitors. Chemical modifications at the C-3 position of BA resulted in compounds, such as dimethylsuccinyl BA (DSB), with various inhibitory activities against human 20S proteasomes. Interestingly, the proteasomal activation by BA and the inhibitory activity of DSB could be abrogated by introducing a side chain at the C-28 position. In summary, this study discovered a class of small molecules that can either activate or inhibit human proteasome activity depending on side chain modifications.
Betulinic acid; proteasome inhibitor; proteasome activator
The 26S proteasome is a cellular proteolytic complex containing 19S regulatory particles and the 20S core proteasome. It was reported that the small molecule b-AP15 targets the proteasome by inhibiting deubiquitination of the 19S regulatory particles of the proteasome complex. An investigation of b-AP15 on the 20S proteasome core suggested that this compound can also inhibit the 20S proteasome with a potency equivalent to that found to inhibit the 19S regulatory particles.
proteasome inhibitor; b-AP15; 19S regulatory particles
Five novel tigliane-type diterpenes, stelleracins A–E (3–7), a novel flavanone dimer, chamaeflavone A (8), and six known compounds were isolated from roots of Stellera chamaejasme. Their structures were elucidated by extensive spectroscopic analyses. The isolated compounds were evaluated for anti-HIV activity in MT4 cells. New compounds 3–5 showed potent anti-HIV activity (EC90 0.00056–0.0068 μM) and relatively low or no cytotoxicity (IC50 4.4–17.2 μM). These new compounds represent promising new leads for development into anti-AIDS clinical trial candidates.
Bevirimat (1, BVM) is an anti-HIV agent that blocks HIV-1 replication by interfering with HIV-1 Gag-SP1 processing at a late stage of viral maturation. However, clinical trials of 1 have revealed a high baseline drug resistance that is attributed to naturally-occurring polymorphisms in HIV-1 Gag. To overcome the drug resistance, 28 new derivatives of 1 were synthesized and tested against compound 1-resistant (BVM-R) HIV-1 variants. Among them, compound 6 exhibited much improved activity against several HIV-1 strains carrying BVM-R polymorphisms. Compound 6 was at least 20-fold more potent than 1 against the replication of NL4-3/V370A, which carries the most prevalent clinical BVM-R polymorphism in HIV-1 Gag-SP1. Thus, compound 6 merits further development as a potential anti-AIDS clinical trial candidate.
Betulinic acid; Bevirimat; HIV-1; Maturation inhibitors; Bevirimat-resistance
Accumulation of aberrant protein aggregates, such as
peptide (Aβ), due to decreased proteasome activities, might
contribute to the neurodegeneration in Alzheimer's disease. In this
study, lithocholic acid derivatives 3α-O-pimeloyl-lithocholic
acid methyl ester (2) and its isosteric isomer (6) were found to activate the chymotrypsin-like activity of
the proteasome at an EC50 of 7.8 and 4.3 μM, respectively.
Replacing the C24 methyl ester in 2 with methylamide
resulted in a complete devoid of proteasome activating activity. Epimerizing
the C3 substituent from an α to β orientation transformed
the activator into a proteasome inhibitor. Unlike the cellular proteasome
activator PA28, proteasome activated by 2 was not inhibited
by Aβ. Furthermore, 2 potently antagonized the
inhibitory effect of Aβ on the proteasome. In summary, compound 2 represents a novel class of small molecules that not only
activates the proteasome but also antagonizes the inhibitory effect
of Aβ on the proteasome.
proteasome activator; lithocholic acid; Alzheimer's
disease; amyloid β
In a continuing study of bevirimat (2), the anti-HIV-maturation clinical trials agent, 28 new betulinic acid (BA, 1) derivatives were designed and synthesized. Among these compounds, 17, with a C-28 MEM ester moiety, and 22, with a C-28 ethyl hexanoate, increased the anti-HIV replication activity compared with 2 by two-fold, while compounds 40–41 and 48–49, with C-28 piperazine or piperidine amide substitutions, increased the activity by three- to fifteen-fold. The best new compound 41 exhibited an anti-HIV IC50 value of 0.0059 μM, compared with 0.087 μM for 2. All of the active compounds showed only anti-maturation effects, as confirmed by TZM-bl assay, in blocking the HIV replication. The results suggest that proper C-28 substitutions can further enhance the anti-maturation activity of 2, without any anti-entry effects. Thus, 41 may serve as a promising new lead for development of anti-AIDS clinical trial candidates.
Twenty-one new 4-substituted diarylaniline compounds (DAANs) (Scheme 2, series 13, 14, and 15) were designed, synthesized, and evaluated against wild-type and drug resistant HIV-1 viral strains. As a result, approximately a dozen new DAANs showed high potency with low nano- to sub-nanomolar EC50 values ranging from 0.2 to 10 nM. The three most promising compounds 14e, 14h, and 15h exhibited high potency against wild-type and drug-resistant viral strains with EC50 values at the sub-nanomolar level (0.29–0.87 nM), and were comparable to or more potent than the new NNRTI drug riplivirine (2) in the same assays. Drug-like physicochemical property assessments revealed that the most active DAANs (EC50 <10 nM) have better aqueous solubility (>1–90 μg/mL at pH 7.4 and pH 2) and metabolic stability in vitro than 2, as well as desirable log P values (<5) and polar surface area (PSA) (<140 Å2). These promising results warrant further development of this novel compound class as potential potent anti-AIDS clinical trial candidates.
Betulinic acid derivatives modified at the C28 position are HIV-1entry inhibitors such as compound A43D; however, modified at the C3 position instead of C28 give HIV-1 maturation inhibitor such as bevirimat. Bevirimat exhibited promising pharmacokinetic profiles in clinical trials, but its effectiveness was compromised by the high baseline drug resistance of HIV-1 variants with polymorphism in the putative drug binding site. In an effort to determine whether the viruses with bevirimat resistant polymorphism also altered their sensitivities to the betulinic acid derivatives that inhibit HIV-1 entry, a series of new betulinic acid entry inhibitors were synthesized and tested for their activities against HIV-1 NL4-3 and NL4-3 variants resistant to bevirimat. The results show that the bevirimat resistant viruses were approximately 5- to10-fold more sensitive to three new glutamine ester derivatives (13, 15 and 38) and A43D in an HIV-1 multi-cycle replication assay. In contrast, the wild type NL4-3 and the bevirimat resistant variants were equally sensitive to the HIV-1 RT inhibitor AZT. In addition, these three new compounds markedly improved microsomal stability compared to A43D.
HIV-1; Entry inhibitor; Maturation inhibitor; Betulinic acid; Berivimat; Berivimat-resistance
Accumulation of aberrant protein aggregates, such as amyloid beta peptide (Aβ), due to decreased proteasome activities might contribute to the neurodegeneration in Alzheimer's disease. In this study, lithocholic acid derivatives 3α-O-pimeloyl-lithocholic acid methyl ester (2) and its isosteric isomer (6) were found to activate the chymotrypsin-like activity of the proteasome at an EC50 of 7.8 and 4.3 μM, respectively. Replacing the C24 methyl ester in 2 with methylamide resulted in a complete devoid of proteasome activating activity. Epimerizing the C3 substituent from an alpha to beta orientation transformed the activator into a proteasome inhibitor. Unlike the cellular proteasome activator PA28, proteasome activated by 2 was not inhibited by Aβ. Furthermore, 2 potently antagonized the inhibitory effect of Aβ on the proteasome. In summary, compound 2 represents a novel class of small molecules that not only activates the proteasome but also antagonizes the inhibitory effect of Aβ on the proteasome.
proteasome activator; lithocholic acid; Alzheimer's disease; amyloid beta
The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4+ peripheral blood mononuclear cells [PBMCs] and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by preexisting cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites.
MicroRNAs (miRNAs) are a ubiquitous class of small regulatory RNAs that serve as posttranscriptional regulators of gene expression. Previous work has suggested that HIV-1 might subvert the function of the cellular miRNA machinery by expressing viral miRNAs or by dramatically altering the level of cellular miRNA expression. Using very sensitive approaches, we now demonstrate that neither of these ideas is in fact correct. Moreover, HIV-1 transcripts appear to largely avoid regulation by cellular miRNAs by adopting an extensive RNA secondary structure that occludes the ability of cellular miRNAs to interact with viral mRNAs. Together, these data suggest that HIV-1, rather than seeking to control miRNA function in infected cells, has instead evolved a mechanism to become largely invisible to cellular miRNA effector mechanisms.
In the present study, a new strategy to link AZT with betulin/betulinic acid (BA) by click chemistry was designed and achieved. This conjugation via a triazole linkage offers a new direction for modification of anti-HIV triterpenes. Click chemistry provides an easy and productive way for linking two molecules, even when one of them is a large natural product. Among the newly synthesized conjugates, compounds 15 and 16 showed potent anti-HIV activity with EC50 values of 0.067 and 0.10 µM, respectively, which are comparable to that of AZT (EC50: 0.10 µM) in the same assay.
Betulin; Betulinic acid; AZT; Anti-HIV; Click chemistry
The current optimization of 2,4-diarylaniline analogs (DAANs) on the central phenyl ring provided a series of new active DAAN derivatives 9a–9e, indicating an accessible modification approach that could improve anti-HIV potency against wild-type and resistant strains, aqueous solubility, and metabolic stability. A new compound 9e not only exhibited extremely high potency against wild-type virus (EC50 0.53 nM) and several resistant viral strains (EC50 0.36 – 3.9 nM), but also showed desirable aqueous solubility and metabolic stability, which were comparable or better than those of the anti-HIV-1 drug TMC278 (2). Thus, new compound 9e might be a potential drug candidate for further development of novel next-generation NNRTIs.
Diarylaniline; NNRTIs; lead optimization; anti-HIV agents
In this study, 1R,2R-dicamphanoyl-3,3-dimethydihydropyrano[2,3-c]xanthen-7(1H)-one (DCX) derivatives were designed and synthesized as novel anti-HIV agents against both wild-type and nonnucleoside reverse transcriptase (RT) inhibitor-resistant HIV-1 (RTMDR-1) strains. Twenty-four DCX analogs (6-29) were synthesized and evaluated against the non-drug-resistant HIV-1 NL4-3 strain, and selected analogs were also screened for their ability to inhibit the RTMDR-1 strain. Compared with the control 2-ethyl-3′,4′-di-O-(-)-camphanoyl-2′,2′-dimethyldihydropyrano[2,3-f]chromone (2-EDCP, 2), one of the best anti-HIV coumarin derivatives in our prior study, three DCX compounds (7, 12, and 22) showed better activity against both HIV strains with an EC50 range of 0.062 – 0.081 μM, and five additional compounds (8, 11, 16, 18, and 21) exhibited comparable anti-HIV potency. Six DCX analogs (7, 11-12, 18, and 21-22) also showed enhanced selectivity index (SI) values in comparison to the control. Structure-activity relationship (SAR) information suggested that the extended conjugated system of the pyranoxanthone skeleton facilitates the interaction of the small DCX molecule within the viral binding pocket, consequently leading to enhanced anti-HIV activity and selectivity. Compared to DCP compounds, DCX analogs are a more promising new class of anti-HIV agents.
1R,2R-dicamphanoyl-3,3-dimethydihydropyrano[2,3-c]xanthen-7(1H)-one (DCX); Anti-HIV activity; Structure-activity relationship (SAR)
Six 3′R,4′R-di-O-(S)-camphanoyl-2′,2′-dimethyldihydropyrano[2,3-f]chromone (DCP) and two 3′R,4′R-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) derivatives were designed, synthesized, and evaluated for inhibition of HIV-1NL4-3 replication in TZM-bl cells. 2-Ethyl-2′-monomethyl-1′-oxa- and -1′-thia-DCP (5a, 6a), as well as 2-ethyl-1′-thia-DCP (7a) exhibited potent anti-HIV activity with EC50 values of 30, 38 and 54 nM and therapeutic indexes of 152.6, 48.0 and 100.0, respectively, which were better than or comparable to those of the lead compound 2-ethyl-DCP in the same assay. 4-Methyl-1′-thia-DCK (8a) also showed significant inhibitory activity with an EC50 of 128 nM and TI of 237.9.
2′-Monomethyl-1′-oxa-DCP; 2′-Monomethyl-1′-thia-DCP; 2-Ethyl-1′-thia-DCP; 4-Methyl-1′-thia-DCK; Anti-HIV activity
In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipophilic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds 13, 20, and 21 showed the best proteasome inhibition activity with IC50 values of 1.42, 1.56, and 1.80 µM, respectively, which are three- to four-fold more potent than the proteasome inhibition controls LLM-F and lactacystin.
In a continuing study of novel anti-HIV agents with drug-like structures and properties, 30 1′-O-, 1′-S-, 4′-O- and 4′-substituted-2′,3′-seco-3′-nor DCP and DCK analogues (8–37) were designed and synthesized. All newly synthesized seco-compounds were screened against HIV-1NL4-3 and a multiple reverse transcriptase (RT) inhibitor-resistant (RTMDR) strain in the TZM-bl cell line, using seco-DCK (7) and 2-ethyl-DCP (4) as controls. Several compounds (14, 18, 19, 22–24, and 32) exhibited potent anti-HIV activity with EC50 values ranging from 0.93 to 1.93 μM and therapeutic index (TI) values ranging from 20 to 39. 1′-O-Isopropoxy-2′,3′-seco-3′-nor-DCP (12) showed the greatest potency among the newly synthesized compounds with EC50 values of 0.47 and 0.88 μM, and TI of 96 and 51, respectively, against HIV-1NL4-3 and RTMDR strains. The seco-compounds exhibited better chemical stability in acidic conditions compared with DCP and DCK compounds. Overall, the results suggested that seco-DCP analogues with simplified structures may be more favorable for development as novel anti-HIV candidates.
2′, 3′-Seco-3′-nor-DCPs; Anti-HIV activity; Structure–activity relationship (SAR)
We prepared 1-(4′-azido-2′-deoxy-2′-fluoro-β -D-arabinofuranosyl)cytosine (10) and its hydrochloride salt (11) as potential antiviral agents based on the favorable antiviral profiles of 4′-substituted nucleosides. Compounds 10 and 11 were synthesized from 1,3,5-O-tribenzoyl-2-deoxy-2-fluoro-D-arabinofuranoside in multiple steps, and their structures were unequivocally established by IR, 1H NMR, 13C NMR, and 19F NMR spectroscopy, HRMS, and X-ray crystallography. Compounds 10 and 11 exhibited potent anti-HIV-1 activity (EC50: 0.3 and 0.13 nM, respectively) without significant cytotoxicity in concentrations up to 100 μM. Compound 11 exhibited extremely potent anti-HIV activity against NL4-3 (wild-type), NL4-3 (K101E), and RTMDR viral strains, with EC50 values of 0.086, 0.15, and 0.11 nM, respectively. Due to the high potency of 11, it was also screened against an NIH Reagent Program NRTI-resistant virus panel containing eleven mutated viral strains and for cytotoxicity against six different human cell lines. The results of this screening indicated that 11 is a novel NRTI that could be developed as an anti-AIDS clinical trial candidate to overcome drug-resistance issues.
4′-Azido-2′-deoxy-2′-fluoro nucleosides; Anti-HIV activity; Nucleoside reverse transcriptase inhibitor (NRTI); Drug resistance
Three novel 1-alkyldaphnane-type diterpenes, stelleralides A–C (4–6), and five known compounds were isolated from the roots of Stellera chamaejasme L. The structures of 4–6 were elucidated by extensive spectroscopic analyses. Several isolated compounds showed potent anti-HIV activity. Compound 4 showed extremely potent anti-HIV activity (EC90 0.40 nM) with the lowest cytotoxicity (IC50 4.3 μM), and appears to be a promising compound for development into anti-AIDS clinical trial candidates.
Based on the structures and activities of our previously identified non-nucleoside reverse transcriptase inhibitors (NNRTIs), we designed and synthesized two sets of derivatives, diarylpyridines (A) and diarylanilines (B), and tested their anti-HIV-1 activity against infection by HIV-1 NL4-3 and IIIB in TZM-bl and MT-2 cells, respectively. The results showed that most compounds exhibited potent anti-HIV-1 activity with low nanomolar EC50 values, and some of them, such as 13m, 14c, and 14e, displayed high potency with subnanomolar EC50 values, which were more potent than etravirine (TMC125, 1) in the same assays. Notably, these compounds were also highly effective against infection by multi-RTI-resistant strains, suggesting a high potential to further develop these compounds as a novel class of NNRTIs with improved antiviral efficacy and resistance profile.
Highly active antiretroviral therapy (HAART) has offered a promising approach for controlling HIV-1 replication in infected individuals. However, with HARRT, HIV-1 is suppressed rather than eradicated due to persistence of HIV-1 in latent viral reservoirs. Thus, purging the virus from latent reservoirs is an important strategy toward eradicating HIV-1 infection. In this study, we discovered that the daphnane diterpene gnidimacrin, which was previously reported to have potent anti-cancer cell activity, activated HIV-1 replication and killed persistently-infected cells at picomolar concentrations. In addition to its potential to purge HIV-1 from latently infected cells, gnidimacrin potently inhibited a panel of HIV-1 R5 virus infection of peripheral blood mononuclear cells (PBMCs) at an average concentration lower than 10 pM. In contrast, gnidimacrin only partially inhibited HIV-1 ×4 virus infection of PBMCs. The strong anti-HIV-1 R5 virus activity of gnidimacrin was correlated with its effect on down-regulation of the HIV-1 coreceptor CCR5. The anti-R5 virus activity of gnidimacrin was completely abrogated by a selective protein kinase C beta inhibitor enzastaurin, which suggests that protein kinase C beta plays a key role in the potent anti-HIV-1 activity of gnidimacrin in PBMCs. In summary, these results suggest that gnidimacrin could activate latent HIV-1, specifically kill HIV-1 persistently infected cells, and inhibit R5 viruses at picomolar concentrations.
In a continued study, 23 3′R,4′R-di-O-(−)-camphanoyl-2′,2′-dimethyldihydropyrano[2,3-f]chromone (DCP) derivatives (5–27) were synthesized, and screened for anti-HIV activity against both a non-drug-resistant NL4-3 strain and multiple reverse transcriptase (RT) inhibitor-resistant (RTMDR-1) strain, using 2-EDCP (4) and 2-MDCP (35) as controls. New DCP analogs 5, 9, 14, and 22 exhibited potent anti-HIV activity against HIVNL4-3 with EC50 and therapeutic index (TI) values ranging from 0.036 μM to 0.14 μM and from 110 to 420, respectively. Compounds 5 and 9 also exhibited good activity against RTMDR-1 (EC50 0.049 and 0.054 μM; TI 310 and 200, respectively), and were two-fold more potent than the leads 4 and 35 (EC50 0.11 and 0.19 μM; TI 60 and 58, respectively). Evaluation of water solubility showed that 5 and 22 were 5–10 times more water soluble than 4. Quantitative structure-activity relationship (QSAR) modeling results were first performed on this compound type, and the models should aid in design of future anti-HIV DCP analogs and potential clinical drug candidates.
3′R,4′R-Di-O-(−)-camphanoyl-2′,2′-dimethyldihydropyrano[2,3-f]chromone (DCP) derivatives; HIV-1; Reverse transcriptase (RT); Structure-activity relationship (SAR)