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. 2020 Mar 19;11(5):766–772. doi: 10.1021/acsmedchemlett.9b00608

5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid Protein

Savina Malancona †,*, Mattia Mori ‡, Paola Fezzardi †, Marisabella Santoriello †, Andreina Basta †, Martina Nibbio †, Lesia Kovalenko §, Roberto Speziale †, Maria Rosaria Battista †, Antonella Cellucci †, Nadia Gennari †, Edith Monteagudo †, Annalise Di Marco †, Alessia Giannini ⊥, Rajhans Sharma §, Manuel Pires §, Eleonore Real §, Maurizio Zazzi ⊥, Maria Chiara Dasso Lang ‡, Davide De Forni , Francesco Saladini ⊥, Yves Mely §, Vincenzo Summa †, Steven Harper †, Maurizio Botta ‡
PMCID: PMC7236274  PMID: 32435383

Abstract

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The HIV-1 nucleocapsid (NC) protein is a small basic DNA and RNA binding protein that is absolutely necessary for viral replication and thus represents a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved sequence offers the opportunity to escape the drug resistance (DR) that emerged following the highly active antiretroviral therapy (HAART) treatment. On the basis of our previous research, nordihydroguaiaretic acid 1 acts as a NC inhibitor showing moderate antiviral activity and suboptimal drug-like properties due to the presence of the catechol moieties. A bioisosteric catechol replacement approach led us to identify the 5-dihydroxypyrimidine-6-carboxamide substructure as a privileged scaffold of a new class of HIV-1 NC inhibitors. Hit validation efforts led to the identification of optimized analogs, as represented by compound 28, showing improved NC inhibition and antiviral activity as well as good ADME and PK properties.

Keywords: Nucleocapsid protein, HIV, NC inhibitors, dihydroxypyrimidine, drug resistance, antiretroviral

AIDS (Acquired Immune Deficiency Syndrome) is still a major global public health issue. According to the Joint United Nation Program on HIV/AIDS (UNAIDS), nearly 38 million people were living with HIV in 2018.1 The current pharmacological strategy relies on the combination of drugs (HAART, highly active antiretroviral therapy) targeting various steps of the viral replication: entry, fusion, reverse transcription, integration, protein maturation. Despite successful in mortality and morbidity reduction, HAART long-term treatment is associated with the insurgence of drug resistance as results of the ability of HIV-1 to mutate.2 In this context, there is an urgent need to develop new drugs to treat HIV infection based on a novel mechanism of action or targets.3 One emerging approach to overcome drug resistance is to target HIV-1 proteins that are highly conserved among phylogenetically distant viral strains and currently not targeted by available therapies. The nucleocapsid (NC) protein of the Human Immunodeficiency Virus type-1 (HIV-1) is a small and basic protein with two zinc fingers that is highly conserved among the retroviruses. This nucleic acid (NA) binding protein is involved in both early and late steps of the HIV-1 replication cycle through its ability to chaperone NAs toward their most stable conformation.4−7 Thus, NC appears as an ideal target for the development of new drugs to prevent HIV-1 replication and complement the highly active antiretroviral therapy.8 Since NC is highly conserved, anti-NC drugs are expected to provide a sustained replication inhibition of the wild type and HIV-1 drug-resistant strains. Three classes of NC inhibitors have been reported based on the mechanism of action: (i) zinc ejectors,9,10 (ii) noncovalent NCIs binding to nucleic acid partners of NC,11,12 and (iii) noncovalent NCIs binding to NC.13,14 The development of NCIs directly binding to NC is currently our main research area, as it is expected to overcome the selectivity and toxicity issues intrinsically related to NCIs belonging to the classes (i and ii). In this work, we describe the most recent advances achieved, leading to the identification of a new class of NCIs showing both good antiviral activity as well as promising in vitro and in vivo profiles.

Recently, we have developed noncovalent NC inhibitors15 by applying a computational structure-based screening of commercial compounds collection followed by in vitro assessment of the NC inhibitory activity. This led to the identification of the natural product nordihydroguaiaretic acid 1 and compound 2 as NC inhibitors sharing a catechol moiety (Figure 1). On the basis of our pharmacophore, the catechol moiety is a common structural feature that plays a critical role in binding NC in a guanidine-like manner by means of the two hydroxyl groups.16−18 Biophysical and computational studies of compound 1 interacting with the protein confirmed the binding postulated by modeling.15 While valuable as a tool compound, the low metabolic profile associated with the catechol group limits the further development of both compounds 1 and 2. The replacement of undesirable functional groups with bioisosters is a common strategy applied in drug discovery to modulate the activity profile, reduce potential toxicities, and circumvent metabolic liabilities.19 In this perspective, using the protocol previously described,16 a virtual screening campaign on the ZINC catalog of commercial products was conducted to identify catechol-like compounds as putative candidate inhibitors of NC. Following virtual screening and clusterization of the top-ranking compounds,15 the dihydroxypyrimidine core scaffold 3 (Figure 1) emerged as a potential replacement for the catechol moiety and thus offered the possibility to develop a new class of NCIs.

Figure 1.

Figure 1

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NC inhibitors based on catechol template 1 and 2. 5,6-Didihydroxypyrimidine 3 as catechol replacement and hit compound 4 active in the NC-inhibition assay. Raltegravir, first in class HIV integrase inhibitor.

Along this line, further scaffold expansion was achieved through the design of a focused virtual library of analogs with the scope of selecting a pool of compounds to be evaluated in vitro for their ability to inhibit the NC chaperone activity. To this aim, a well-established fluorescence assay20 (NC-inhibition assay) was used; it allows researchers to monitor the destabilization of cTAR DNA, the complementary sequence to the transactivation response element of the HIV-1 genome labeled with the Alexa488 dye and Dabcyl quencher.21,22 The protein concentration used in the inhibition assay is 1 μM, and thus, 0.5 μM is the lowest IC50 value measurable. This effort conducted to the identification of compound 4 as a positive hit (Figure 1), showing an IC50 below 200 μM (Table 1). Despite its modest potency, compound 4 represented an optimal starting point from the development perspective due to the favorable drug-like properties of the dihydroxypyrimidine carboxamide class. This class of compounds is known to have led to the discovery of Raltegravir23 (Figure 1), the first in class HIV integrase inhibitor. Furthermore, in the antiviral field, the dihydroxypyrimidine core has been investigated to target other NA processing enzymes such as HIV-1 RT24 or HCV NS5B RNA-dependent RNA polymerase.25 Because of the close structural similarity between compound 4 and Raltegravir, a possible polypharmacology profile could be expected in the development of this class of compounds as NC inhibitors. Nonetheless, first rough structural comparison and lack of activity of Raltegravir in the NC inhibition assay (data not shown) suggested that a degree of selectivity was already present in the hit compound 4. The structure activity relationship (SAR) of the dihydroxypyrimidine carboxamide as inhibitors of the HIV-integrase enzyme has been fully elucidated and well documented in the literature.26−29 Optimized HIV-integrase inhibitors show a marked preference for the N-methylpyrimidone core, benzylic carboxamide, and neutral aryl or alkyl substituent at the 2-position of the pyrimidine core. In contrast, the catechol-like dihydroxypyrimidine core of the hit compound 4 is decorated with an alkyl amide that in the HIV integrase inhibitor completely abolishes the activity against this target, and a basic aromatic substituent in the 2-position of the pyrimidine.28 A hit validation and optimization process was thus conducted on 4 with the aim of improving the potency as NC inhibitor. The compounds were also tested for cell-based antiretroviral activity. The in vitro and in vivo ADME properties were finally evaluated for the most interesting analogs.

Table 1. In Vitro NC Inhibition for the Analogs Exploring the Amide and Aromatic Substituent Regions.

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a

Data are average ± SD of a least two independent experiments.

b

N.A. = not active.

c

IC40.

d

IC35.

While retaining the central dihydroxypyrimidine core, structural modifications were introduced first in the aromatic region at the 2-position (R2, Table 1). The 2-(pyridin-2-yl) group emerged as the privileged substitution for the activity: the replacement with a simple phenyl 5 or replacement with the 2-(pyridin-3-yl) or 2-(pyridin-4-yl) isomers, respectively compounds 6 and 7, resulted in complete loss of the activity. Neither a 2-phenethyl, 2-(1-phenylethyl), nor bulky 2-(tert-butyl) substituents were tolerated, compounds 8, 9, and 10, respectively. The amide moiety (R1, Table 1) of compound 4 is believed from modeling to be oriented toward the hydrophobic binding site of the protein. The SAR in the amide region showed that the replacements of the N-(cyclohexylmethyl) group with N-benzyl 11, N-ethyl 12, and the tertiary amide 13 are not tolerated. The exploration of the linker length pointed out that anchoring the cyclohexyl directly to the amide nitrogen as for 14 produces a two-fold loss of activity in the NC inhibition assay with respect to the hit compound 4, whereas the elongation of the linker by one carbon atom 15 is beneficial for the activity and accounts for a four-fold improved activity. Similar gain of potency is achieved with the branched analogs (R)-N-(1-cyclohexylethyl) 16 and (S)-N-(1-cyclohexylethyl) 17, showing the same activity regardless of chirality. The results obtained are in line with the proposed binding mode that suggests the amide occupying the hydrophobic pocket and that the lipophilic substituents are thus well tolerated, chirality is not influencing conformation and binding of the amide moiety.

Preliminary SAR at the 2-position of the 5,6-dihydroxypyrimidine core has shown that 2-(pyridin-2-yl) substituent is required for the activity. Further optimization was thus conducted introducing substituents on the pyridine ring. First modifications introduced were the methyl (18–22) and trifluoromethyl (23–26) groups, which were selected considering the differences in the electron donating and withdrawing ability as well as lipophilicity. Regardless of the electronic nature, the introduction of a substituent in positions 4 and 5 is beneficial for the activity, affording compounds 20 and 25 showing IC50s around 10 μM. In the other positions of the pyridine ring, two-fold activity gain was achieved with respect to the hit 4. A variety of other substituents were evaluated, in particular, the most relevant results were obtained with the introduction of 5-methoxy and 5-chlorine groups on the pyridine ring (respectively, compounds 27 IC50 = 20 μM and 28 IC50 = 2 μM). To complete the exploration at the 2-position of the pyrimidine core, a series of heterocycles retaining the 2-nitrogen atom present in the 2-pyridine substituent was explored. Best results were achieved with fused bicyclic groups, such as the quinoline analogs 29, showing more than 40-fold improvement in the NC inhibition with respect to the original compound 4. Smaller 5-member ring heterocycles such as thiophene or thiazole were not tolerated (data not shown).

Modification at the dihydroxypyrimidine core showed that both hydroxyl groups are essential for the activity. Indeed, compounds 30 and 31 lacking, respectively, the 4- and 5-hydroxyl substituent, were found inactive in the NC inhibition assay (Figure 2).

Figure 2.

Figure 2

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Dihydroxypyrimidine core modifications leading to lack of NC inhibition.

The 5,6-dihydroxypyrimidine core exists in two tautomeric forms, namely dihydroxypyrimidine and pyrimidone. A different hydrogen bond donator/acceptor asset, potentially able to influence the binding to the nucleocapsid protein, is associated with each form. The N-methyl pyrimidone analog 32 resulted to be completely inactive in the NC inhibition assay, indicating the catechol form as decisive for NC inhibition, in contrast to the preference for the pyrimidinone form in the HIV-1 integrase area. The SAR conducted on the hit compound 4 has shown that it is possible to modulate NC activity and improve 80-fold its potency (cmpd 28), thus obtaining a number of compounds in the low micromolar range of activity.

The binding mode of most potent NC inhibitors identified was investigated by molecular docking simulations and reported in Figure 3 for compound 28 and Figure S1 for compounds 4, 20, and 25. To this aim, we took advantage of the computational protocol already refined by our research group for the NC protein and based on molecular docking with the FRED program (OpenEye) against the NMR structure of the NC in complex with a small molecule inhibitor (PDB: 2M3Z), see also the Supporting Information.15,17,16 Overall, the molecules shared a highly similar binding mode and proved to fit the hydrophobic pocket of the NC. Besides a π-stacking interaction with the side chain of Trp37, the core dihydroxypyrimidine established H-bonds with the backbone of Gly35, and Met46, and with the side-chain of Gln45. In addition, the amide moiety established hydrophobic interactions with the side chain of residues from the NC hydrophobic platform17 such as Phe16, Ala25, Trp37, and Met46 (Figure 3). Overall, this binding mode is highly consistent with the binding mode of NA nucleotides30 and of nordihydroguaiaretic acid 1.15 Most notably, the predicted binding for these ihydroxypyrimidine also overlaps with the binding mode of the NC inhibitor characterized by NMR spectroscopy in complex with the NC, whose structure was used as a receptor in molecular docking simulation31 (Supporting Information, Figure S2). A good correlation between the score of compounds 4, 20, 25, and 28 and their −logIC50 was observed (R2 = 0.939) (Supporting Information, Figure S3). Finally, the binding mode described above and shown in Figure 3 and Figure S1 clearly accounts for the lack of NC inhibition by derivatives deprived of the dihydroxyl moiety, such as 30 and 31, or bearing the N-methyl pyrimidone substructure such as 32.

Figure 3.

Figure 3

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Predicted binding mode of 28 within the hydrophobic pocket of the NC. The protein is shown as a green cartoon. Residues within 5 Å from the ligands are shown as lines and are labeled. Compound 28 is shown as yellow sticks. H-bond interactions are highlighted by black dashed lines.

The antiviral activity on the whole replication cycle of HIV-1 was evaluated for selected compounds by means of the cell-based assay named BiCycle (Table 2). The assay consists of a first infection round of the T-cell line MT-2 using HIV-1 wild-type reference strain NL4–3, followed by a second infection round of the reporter cell line TZM-bl.32 Hit compound 4 shows activity in the low micromolar range. A similar activity is found for the analog 15, which is more active in the biochemical assay as result of the amide elongation.

Table 2. Antiviral and Cytotoxicity Profilea.

  BiCycle Wild type strain NL4–3 Hela
 PBMC MTS
 Strain 11808 (PI)f Strain 7401 (NRTI)f Strain 12231 (NNRTI)f Strain 11845 (INI)f
ID EC50 (μM)b CC50 (μM)b SI-Helac CC50 (μM)b SI-PBMCd EC50 (μM)b EC50 (μM)b EC50 (μM)b EC50 (μM)b
4 1.2 ± 0.7 3.6 ± 0.7 3 2.6 ± 1.7 2        
15 2.3 ± 2 4.2 ± 3.4 2 11 ± 6.0 5        
16 0.7 ± 0.1 3.2 ± 0.9 5 3.0 ± 1.4 4        
17 0.2 ± 0.1 0.6 ± 0.04 3 2.4 ± 1 12        
20 0.2 ± 0.1 2.7 ± 0.4 13 3.1 ± 0.8 16        
25 0.4 ± 0.3 6.0 ± 0.2 15 1.1 ± 0.1 3        
27 0.04 ± 0.03 3.0 ± 0.1 75 2.7 ± 0.1 75 0.056 ± 0.02 (1.4)e 0.031 ± 0.02 (0.8)e 0.11 ± 0.04 (2.8)e 0.09 ± 0.04 (2.3)e
28 0.1 ± 0.03 3.0 ± 0.5 30 2.5 ± 0.2 25 0.02 ± 0.01 (0.2)e 0.01 ± 0.005 (0.1)e 0.08 ± 0.06 (0.8)e 0.07 ± 0.08 (0.7)e
29 0.1 ± 0.05 2.1 ± 0.3 21 5 ± 2 50 0.16 ± 0.1 (1.6)e 0.13 ± 0.08 (1.3)e 0.21 ± 0.07 (2.0)e 0.09 ± 0.02 (0.9)e
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a

Susceptibility to viral strains harbouring resistance to drug used in clinical practice.

b

Data are average ± SD for a least two independent experiments.

c

SI-Hela: HELA CC50/BiCycle EC50.

d

SI-PBMC: PBMC CC50/BiCycle EC50.

e

Fold change values indicate the ratio between IC50 values from drug-resistant and NL4–3 wild type reference strains.

f

NIH AIDS Reagent Program catalogue number of resistant strains (www.aidsreagent.org). PI, resistance to protease inhibitor; NRTI, resistance to nucleoside reverse transcriptase inhibitor; NNRTI, resistance to non-nucleoside reverse transcriptase inhibitor; INI, resistance to integrase inhibitor.

A beneficial effect on the antiviral activity is seen when the branched amide is present, with both 16 and 17 enantiomers active in the submicromolar range. The difference between the R and S enantiomers in term of antiviral activity is very modest (3-fold), confirming the behavior observed in the NC inhibition assay. Analogs substituted in the aromatic region such as 20, 25, 27, 28, and 29 exhibit nanomolar antiviral activity with EC50s ranging from 40 to 400 nM. In parallel, cytotoxicity was evaluated in Hela and peripheral blood mononuclear cells (PMBC), and the selectivity indexes (SI) were calculated as the ratio between the CC50 and EC50 values measured in their respective assays (see footnote of Table 2). Despite the narrow difference between the activity and cytotoxicity found for 4 and close analogues (SI range 2–5-fold), much higher SI values were found for compounds 20, 27, 28, and 29 as a result of their improved activity in cells (SI in the range 13–75). Overall, these data clearly show that the potency and selectivity can be modulated and improved in future investigations of the series. The inhibition of NC is expected to overcome drug resistance. To prove this hypothesis, most potent compounds were tested against a representative panel of drug-resistant HIV-1 strains (Table 2). Notably, only a 1–2-fold IC50 shift was measured compared to wild type IC50, consistent with the hypothesis that NC inhibitors retain their full potency against resistant strains.

To further characterize the new class of NC inhibitors (NCIs), a series of in vitro and in vivo tests was performed on a small set of selected compounds. The metabolic stability was evaluated in vitro in rat and human species, plasma, and hepatocytes matrices (Table 3). Data show that compounds 25, 27, 28, and 29 were stable up to 1 h or more in both plasma and hepatocytes matrices. The same compounds were not inhibitors of major human cytochrome P450 isoforms (CYP1A2, CYP2D6, CYP3A4) and the hERG ion channels. On the basis of the potency profile in the different assays and the in vitro DMPK data, compounds 27, 28 and 29 were selected for in vivo PK studies in C57BL/6 mice. Compounds were administrated IV and PO at 2 and 5 mg/kg, respectively. Eight time points were taken up to 24 h to obtain a plasma PK profile (Table 4).

Table 3. In Vitro Profiling for Compounds 25, 27, 28, and 29.

  in vitro profiling
ID mouse plasma t1/2 (h) human plasma t1/2 (h) mouse hepatocytes t1/2 (h) human hepatocytes t1/2 (h) hERG IC50 (μM) CYP1A2 IC50 (μM) CYP2D6 IC50 (μM) CYP3A4 IC50 (μM)
25 >1 >1 2.4 >4 >30 >30 >30 >30
27 >24 >6 1.9 >4 >30 >30 >30 >30
28 >6 >6 1.7 >4 >30 >30 >30 >30
29 3.7 ND 1.7 2.6 >30 >30 >30 >30
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Table 4. PK Parameters for Compounds 27, 28, and 29 Following i.v. and p.o. Administration in C57BL/6 Mice at 2 mg/kg and 5 mg/kg, Respectively.

  in vivo profiling
ID t1/2 (h) Vdss (L/kg) Cl (mL/min/kg) %F
25        
27 1.4 0.8 33.1 25
28 3.1 1.2 37.1 40
29 1.1 0.8 30.2 26
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Compounds 27 and 29 showed similar PK parameters: medium-low plasma clearance (33 and 30 mL/min/kg, respectively) and medium volume of distribution (Vdss 0.8 L/kg for both) with a corresponding half-life of 1.4 and 1.1 h, respectively, for 27 and 29. The oral bioavailability was good (25% and 26%, respectively). Compared to 27 and 29, compound 28 showed a better profile presenting higher oral bioavailability (40%), prolonged half-life of 3.1 h, and volume of distribution of 1.2 L/kg.

To assess a preliminary polypharmacology profile, compounds 4, 27, and 29 were tested for their ability to inhibit the HIV Integrase enzyme by means of the well-established LEDGF-dependent IN activity assay33 resulting not active up to 10 uM, Raltegravir used as positive control showed IC50= 0.027 ± 0.01 μM.

In summary, based on our previous studies, the catechol moiety is a privileged structure to achieve NC inhibition as demonstrated by compound 1. In the prospective of drug development, the intrinsic metabolic liability of the catechol group is an issue to be considered. To overcome this issue, we decided to search for catechol-like scaffold with improved physicochemical properties able to bind NC protein. The process allowed the identification of the 5,6-dihydroxypyrimidine-4-carboxamide analog 4 as representative of a new class of HIV-1 nucleocapsid inhibitors. Initial SAR studies were conducted to prove the bona fide nature of the hit and identify the key structural features needed to achieve and modulate NC inhibition. Optimized analogs such as 27, 28, and 29 not only showed nanomolar antiviral activity and improved selective index but also were active against a panel of HIV-1 resistant strains in agreement with the proposed mechanism of action. Furthermore, the compounds showed a very clean profile against the major CYP-P450 isoforms and the hERG channel. The pharmacokinetic profile in mice was good for all three compounds but in particular for compound 28, which shows 40% oral bioavailability, good half-life, and moderate clearance. In conclusion, we identified an unprecedented structural class of NC inhibitors with good drug-like properties. In particular, compound 28 represents a novel NC inhibitor lead with low nanomolar potency in cell-based assay, low cytotoxicity, and good PK profile. It represents a good starting point for further characterization and optimization.

Acknowledgments

We acknowledge Fabrizio Colaceci (in vivo work); Costanza Iaccarino and Letizia Lazzaro (compounds purification and QC); and Prof. Enzo Tramontano and Francesca Esposito Dipartimento di Scienze della Vita e dell’Ambiente Università di Cagliari for the HIV-IN assay. The authors wish to thank the OpenEye Free Academic Licensing Program for providing a free academic license for molecular modeling and chemoinformatics software. YM is grateful to the Institut Universitaire de France (IUF) for support and providing additional time to be dedicated to research.

Glossary

Abbreviations

AIDS

acquired immune deficiency syndrome

HAART

highly active antiretroviral therapy

NC

nucleocapsid protein

HIV-1

human immunodeficiency virus type-1

NCIs

nucleocapsid protein inhibitors

HIV-IN

human immunodeficiency virus integrase

SI

selectivity index

SAR

structure–activity relationship

NMR

nuclear magnetic resonance

PK

pharmacokinetics

hERG

human Ether-à-go-go-related gene

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00608.

  • Synthetic experimental details and characterization data, description of biological assay protocols, modeling (PDF)

Author Present Address

# AB, Tubilux Pharma spa, via Costarica 20, 00071 Pomezia, Rome, Italy.

Author Present Address

∥ VS, Department of Pharmacy, University of Naples “Federico II”, Via D. Montesano 49, 80131 Naples, Italy.

Author Contributions

□ These authors equally contributed to this work. All authors have given approval to the final version of the manuscript.

This project has received funding from the European Union’s Seventh Programme for research, technological development, and demonstration under Grant Agreement No. 601969.

The authors declare no competing financial interest.

Dedication

△ This work is dedicated to the beloved memory of Prof. Maurizio Botta (August 2, 2019) and Steven Harper (June 30, 2019).

Supplementary Material

ml9b00608_si_001.pdf (714.4KB, pdf)

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