molecules

Review CCR5/CXCR4 Dual Antagonism for the Improvement of HIV Therapy

Fedora Grande 1,* , Maria Antonietta Occhiuzzi 1, Bruno Rizzuti 2,* , Giuseppina Ioele 1, Michele De Luca 1 , Paola Tucci 1 , Valentina Svicher 3, Stefano Aquaro 1,† and Antonio Garofalo 1,† 1 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Ampliamento Polifunzionale, Via P. Bucci, 87036 Rende (CS), Italy; [email protected] (M.A.O.); [email protected] (G.I.); [email protected] (M.D.L.); [email protected] (P.T.); [email protected] (S.A.); [email protected] (A.G.) 2 CNR-NANOTEC, Licryl-UOS Cosenza and CEMIF.Cal, Department of Physics, University of Calabria, Via P. Bucci, 87036 Rende (CS), Italy 3 Department of Experimental Medicine, University of Rome “Tor Vergata”, 00133 Rome, Italy; [email protected] * Correspondence: [email protected] (F.G.); [email protected] (B.R.); Tel.: +39-098-449-3019 (F.G.); +39-098-449-6078 (B.R.) † These authors contributed equally to the present work.

 Academic Editor: Stefano Aquaro  Received: 15 January 2019; Accepted: 1 February 2019; Published: 2 February 2019 Abstract: HIV entry in the host requires the interaction with the CD4 membrane receptor, and depends on the activation of one or both co-receptors CCR5 and CXCR4. Former selective co-receptor antagonists, acting at early stages of infection, are able to impair the receptor functions, preventing the viral spread toward AIDS. Due to the capability of HIV to develop resistance by switching from CCR5 to CXCR4, dual co-receptor antagonists could represent the next generation of AIDS prophylaxis drugs. We herein present a survey on relevant results published in the last few years on compounds acting simultaneously on both co-receptors, potentially useful as preventing agents or in combination with classical anti-retroviral drugs based therapy.

Keywords: receptors; dual drugs; HIV entry; AIDS

1. Introduction The most successful treatment of patients infected with HIV-1 (human immunodeficiency 1) resides so far in the administration of a combination of antiretroviral agents targeting specific proteins necessary to the viral replication. Such a therapeutic strategy, known as highly active antiretroviral therapy (HAART) [1,2], has shown to be particularly effective during the progression of the full-blown viral infection, whereas the same approach was less applicable in the early stages of the infection when infected people are not warned by specific symptoms. The most common agents included in HAART are inhibitors of viral entry and fusion, or drugs targeting , integrase, and protease . An alternative strategic approach focuses on a multi-target inhibition of the viral entry, before a full spreading of the virus into host cells. The viral entry consists of a complex sequence of events mediated by several viral/cellular membrane proteins, with the interaction between the viral glycoprotein gp120 and CD4 cellular receptor constituting an essential initial step. A further interaction of such glycoprotein with any of the two co-receptors CC- 5 (CCR5) and CXC-chemokine receptor 4 (CXCR4) is also needed to promote appropriate conformational modifications during such an early stage of the viral cycle [3].

Molecules 2019, 24, 550; doi:10.3390/molecules24030550 www.mdpi.com/journal/molecules Molecules 2019, 24, 550 2 of 11

Molecules 2019, 24, x 2 of 11 Both CCR5 and CXCR4 are G-protein coupled chemokine receptors determining HIV-1 cellular tropism.primary Incell particular, infection, being this preferentially co-receptor utilizing mainly CCR5 expressed (R5 strains) on mac arerophages. responsible On the for theother primary hand, cellthe infection, viral replication being this capacity co-receptor is attributed mainly expressed to viruses on preferentially macrophages. interacting On the other with hand, CXCR4 theviral (X4 replicationstrains), being capacity this is second attributed co-receptor to viruses mainly preferentially expressed interacting on T-lymphocytes with CXCR4. Finally, (X4 strains), dual-tropic being thisviruses second are defined co-receptor as those mainly usin expressedg both co on-recepto T-lymphocytes.rs (Figure Finally,1) [4,5]. dual-tropic viruses are defined as those using both co-receptors (Figure1)[4,5].

Figure 1. Schematic representation of chemokine receptors mediating HIV cells entry.

WhereasFigure a large 1. S numberchematic of representation agents acting of selectively chemokine on receptors either CCR5 mediating or CXCR4 HIV cells have entry. been described in the literature [6,7], only a few dual antagonists have been so far identified, suggesting that it is possibleWhereas to simultaneously a large number target of the agents two acting proteins selectively due to their on similaritieseither CCR5 in or the CXCR4 overall have structures been anddescribed in the in binding the literature site shapes. [6,7], In only fact, a chemokinefew dual antagonists receptors havehave abeen common so far molecular identified, architecture, suggesting whichthat it isis conservedpossible to within simultaneously the family target of G protein-coupledthe two proteins receptors due to their (GPCRs). similarities As members in the overall of this uniquestructures family, and CCR5in the andbinding CXCR4 site share shapes a structure. In fact, chemoki similarityne (Figure receptors2, top), have such a common as the presence molecular of sevenarchitecture, transmembrane which is domains.conserved Both within receptors the family consist of ofG 352protein amino-coupled acids withreceptors a high (GPCRs). number As of prolinemembers residues, of this unique a C-terminal family, threonine CCR5 and and CXCR4 serine-rich share a cytoplasmic structure similarity region, four (Figure extracellular 2, top), such loops as richthe presence in cysteines of seven and an transmembrane N-terminal extracellular domains. domainBoth receptors [6,8]. In consist particular, of 352 overlapping amino acid regionss with a of high the bindingnumber pockets of proline of CCR5 residues and CXCR4, a C-terminal (Figure2 , threonine bottom) may and contribute serine-rich to allowcytoplasmic these two region, proteins four to Molecules 2019, 24, x 3 of 11 hostextracellular the same loops , rich although in cysteine not necessarilys and an inN- theterminal same binding extracellular mode and domain with [6,8 an identical]. In particular, affinity. overlapping regions of the binding pockets of CCR5 and CXCR4 (Figure 2, bottom) may contribute to allow these two proteins to host the same ligand, although not necessarily in the same binding mode and with an identical affinity.

Figure 2. Cont.

Figure 2. Similarities in the overall structures and binding pockets of CCR5 and CXCR4. Structures of complexes are taken from the Protein Data Bank entry 6AKX (magenta) [9] and 3ODU (cyan) [10] for CCR5 and CXCR4, respectively, and superimposed by least squares fit on protein Cα atoms. (Top) Overall structure of (A) CCR5, (B) both receptors, and (C) CXCR4; (bottom) detail of the binding site of (D) CCR5, (E) both receptors, and (F) CXCR4. The ligands bound to CCR5 and CXCR4 (the 1- heteroaryl-1,3-propanediamine derivative A4R and the antagonist IT1t, respectively) are shown with atoms in standard colours (C, grey; N, blue; O, red; S, yellow) in panels A, C, D and F, and with C atoms in the same colour as their receptor (magenta for A4R, and cyan for IT1t) in panels B and E.

Due to the marked similarities between the active sites of CCR5 and CXCR4, many ligands could be recognized by both receptors. However, RANTES, also known as chemokine ligand 5 (CCL5), was proven to be the natural ligand selective for CCR5 [11], whereas stromal cell derived factor 1 (SDF1) is the natural ligand of CXCR4 [12]. It is well known that the multi-step HIV entry process is not only mediated by the interaction between the viral gp120 with the cellular receptor CD4, but it also requires the activation of one or two chemokine co-receptors. Among these, CCR5 and CXCR4 exhibit this crucial role. The capability of HIV to use either CCR5 or CXCR4, or both at the same time, determines the viral tropism, since, as already stated. In particular, virus strains utilizing CXCR4 or both co-receptors are associated with a higher incidence of AIDS development [13]. From all these considerations, the challenge to discover new CCR5 and CXCR4 antagonists is of paramount importance to improve the therapeutic armamentarium against HIV infection, and the possibility to develop dual co-receptor antagonists appears particularly appealing.

2. CCR5 and CXCR4 Antagonists

2.1. Earlier Co-Receptor Antagonists Nowadays, the choice for the clinical use of a CCR5 or CXCR4 antagonists is suggested by the identification of viral strain tropism in patients. A rapid resistance onset represents however a limit, since the inhibition of a single co-receptor drives the virus to shift to the more virulent dual tropic strain [14,15]. Among selective and efficacious CCR5 antagonists, (Figure 3, compound 1) appeared as the most intriguing compound, being the only one already approved by the Food and Drug Administration (FDA), endowed with a favorable resistance profile. On the other hand, Plerixafor (or AMD-3100) (Figure 3, compound 2) appeared as the most useful CXCR4 antagonist identified during a research program on anti-HIV agents. Although this compound is clinically used

Molecules 2019, 24, x 3 of 11

Molecules 2019, 24, 550 3 of 11

Figure 2. Similarities in the overall structures and binding pockets of CCR5 and CXCR4. Structures Figureof complexes2. Similarities are takenin the from overall the Proteinstructures Data and Bank binding entry 6AKX pockets (magenta) of CCR5 [9 ]and and CXCR4. 3ODU (cyan) Structures [10] of complexesfor CCR5 are and taken CXCR4, from respectively, the Protein and Data superimposed Bank entry by6AKX least (magenta) squares fit on[9] protein and 3ODU Cα atoms. (cyan) (Top) [10] for CCR5Overall and CXCR4, structure respectively, of (A) CCR5, and (B) bothsuperimposed receptors, andby least (C) CXCR4; squares (bottom) fit on protein detail of Cα the atoms. binding (Top) Overallsite structure of (D) CCR5, of (A (E)) CCR5, both receptors, (B) both and receptors, (F) CXCR4. and The(C) CXCR4; ligands bound (bottom to) CCR5 detail and of the CXCR4 binding (the site of (D1-heteroaryl-1,3-propanediamine) CCR5, (E) both receptors, and derivative (F) CXCR4. A4R and The the antagonistligands bound IT1t, respectively) to CCR5 and are shownCXCR4 with (the 1- heteroarylatoms- in1,3 standard-propanediamine colours (C, derivative grey; N, blue; A4R O, and red; the S, yellow)antagonist in panels IT1t, respectively) A, C, D and F, are and shown with C with atoms in the same colour as their receptor (magenta for A4R, and cyan for IT1t) in panels B and E. atoms in standard colours (C, grey; N, blue; O, red; S, yellow) in panels A, C, D and F, and with C atomsDue in the to the same marked colour similarities as their receptor between (magenta the active for sites A4R, of and CCR5 cyan and for CXCR4, IT1t) in many panels ligands B and couldE. be recognized by both receptors. However, RANTES, also known as chemokine ligand 5 (CCL5), wasDue provento the marked to be the similarities natural ligand between selective the foractive CCR5 sites [11 of], whereasCCR5 and stromal CXCR4 cell, many derived ligands factor could 1 be recognized(SDF1) is the by natural both receptors. ligand of CXCR4However, [12]. RANTES, It is well knownalso known that the as multi-step chemokine HIV ligand entry 5 process (CCL5), is was provennot to only be mediatedthe natural by ligand the interaction selective between for CCR5 the viral[11], gp120whereas with stromal the cellular cell derived receptor factor CD4, but 1 (SDF1 it ) is thealso natural requires ligand the activationof CXCR4 of [12] one. It or is two well chemokine known that co-receptors. the multi Among-step HIV these, entry CCR5 process and CXCR4 is not only mediatedexhibit by this the crucial interaction role. The between capability the of HIV viral to use gp120 either with CCR5 the or cellular CXCR4, or receptor both at the CD4, same but time, it also requiresdetermines the activation the viral of tropism, one or two since, chemokine as already stated.co-receptors. In particular, Among virus these, strains CCR5 utilizing and CXCR4 CXCR4 exhibit or this both crucial co-receptors role. The are capability associated of with HIV a higher to use incidence either CCR5 of AIDS or development CXCR4, or [13 both]. at the same time, From all these considerations, the challenge to discover new CCR5 and CXCR4 antagonists is of determines the viral tropism, since, as already stated. In particular, virus strains utilizing CXCR4 or paramount importance to improve the therapeutic armamentarium against HIV infection, and the both co-receptors are associated with a higher incidence of AIDS development [13]. possibility to develop dual co-receptor antagonists appears particularly appealing. From all these considerations, the challenge to discover new CCR5 and CXCR4 antagonists is of paramount2. CCR5 importance and CXCR4 to Antagonists improve the therapeutic armamentarium against HIV infection, and the possibility to develop dual co-receptor antagonists appears particularly appealing. 2.1. Earlier Co-Receptor Antagonists 2. CCR5 andNowadays, CXCR4 the Antagonists choice for the clinical use of a CCR5 or CXCR4 antagonists is suggested by the identification of viral strain tropism in patients. A rapid resistance onset represents however a limit, 2.1. Earliersince the Co inhibition-Receptor Antagonists of a single co-receptor drives the virus to shift to the more virulent dual tropic strain [14,15]. Among selective and efficacious CCR5 antagonists, Maraviroc (Figure3, compound 1) appearedNowadays, as thethe most choice intriguing for the compound,clinical use being of a CCR5 the only or one CXCR4 already antagonist approveds byis suggested the Food and by the identificationDrug Administration of viral strain (FDA), tropism endowed in patients. with a favorable A rapid resistance resistance profile. onset On represents the other hand, however Plerixafor a limit, since(or the AMD-3100) inhibition (Figureof a single3, compound co-receptor2) appeared drives the as thevirus most to usefulshift to CXCR4 the more antagonist virulent identified dual tropic strainduring [14,15 a]. research Among programselective on and anti-HIV efficacious agents. CCR5 Although antagonists, this compound Maraviro isc clinically (Figure used3, compound for the 1) appearedmobilization as the ofmost hematopoietic intriguing stemcompound cells in, being patients, the only it one is not already yet approved approved in the by treatment the Food of and DrugHIV Administration infection due to ( itsFDA severe), endowed with [16, 17 a]. favorable resistance profile. On the other hand, PlerixaforIn (or particular, AMD- AMD31003100) (Figure was shown3, compound to inhibit HIV-12) appeared entry into as the the target most cell useful by specifically CXCR4 bindingantagonist to CXCR4 (and not to any other receptor of either CXC- or CC-). Previous studies had identified during a research program on anti-HIV agents. Although this compound is clinically used demonstrated that AMD3100 was very active against a broad range of T-tropic HIV-1 and HIV-2 strains at a low nanomolar concentrations [18], and poorly active against M-tropic strains [19]. Furthermore, AMD3100 was shown to inhibit binding of the CXC-chemokine (SDF-1alpha) to CXCR4 and subsequent signal transduction, thus preventing also the functioning of CXCR4 as CXC-chemokine receptor [20]. Molecules 2019, 24, x 4 of 11

for the mobilization of hematopoietic stem cells in cancer patients, it is not yet approved in the treatment of HIV infection due to its severe side effects [16,17]. In particular, AMD3100 was shown to inhibit HIV-1 entry into the target cell by specifically Molecules 2019, 24, x 4 of 11 binding to CXCR4 (and not to any other receptor of either CXC- or CC-chemokines). Previous studies had demonstrated that AMD3100 was very active against a broad range of T-tropic HIV-1 and HIV- for the mobilization of hematopoietic stem cells in cancer patients, it is not yet approved in the 2 strains at a low nanomolar concentrations [18], and poorly active against M-tropic strains [19]. treatment of HIV infection due to its severe side effects [16,17]. Furthermore, AMD3100 was shown to inhibit binding of the CXC-chemokine (SDF-1alpha) to CXCR4 In particular, AMD3100 was shown to inhibit HIV-1 entry into the target cell by specifically and subsequent signal transduction, thus preventing also the functioning of CXCR4 as CXC- binding to CXCR4 (and not to any other receptor of either CXC- or CC-chemokines). Previous studies chemokine receptor [20]. had demonstrated that AMD3100 was very active against a broad range of T-tropic HIV-1 and HIV- In 2004, AMD3451 (Figure 3, compound 3) was reported as the first dual antagonist of CCR5 and 2 strains at a low nanomolar concentrations [18], and poorly active against M-tropic strains [19]. CXCR4 [21]. Although its chemical structure resembles the one of the CXCR4 selective inhibitor Furthermore, AMD3100 was shown to inhibit binding of the CXC-chemokine (SDF-1alpha) to CXCR4 AMD3100, it was found to be active also against the CCR5 co-receptor. Its moderate potency did not Moleculesand subsequent2019, 24, 550 signal transduction, thus preventing also the functioning of CXCR4 as CXC4 of 11- allow to carry on a proper clinical investigation, but its identification spurred researchers toward the chemokine receptor [20]. development of more effective dual-tropic CCR5/CXCR4 antagonists [22]. In 2004, AMD3451 (Figure 3, compound 3) was reported as the first dual antagonist of CCR5 and CXCR4 [21]. Although its chemical structure resembles the one of the CXCR4 selective inhibitor AMD3100, it was found to be active also against the CCR5 co-receptor. Its moderate potency did not allow to carry on a proper clinical investigation, but its identification spurred researchers toward the development of more effective dual-tropic CCR5/CXCR4 antagonists [22].

Figure 3. Chemical structures of Maraviroc (1), Plerixafor (2), and AMD3451 (3). Figure 3. Chemical structures of Maraviroc (1), Plerixafor (2), and AMD3451 (3). In 2004, AMD3451 (Figure3, compound 3) was reported as the first dual antagonist of CCR5 and CXCR4About ten[21 ]. years Although later itsit was chemical proven structure that Maraviroc resembles, initially the one identified of the CXCR4 as a selectiveselective inhibitor CCR5 inhibitor, was also an effective therapeutic alternative against dual mixed tropic HIV strains [23]. AMD3100, it was found to be active also against the CCR5 co-receptor. Its moderate potency did not allowAntiviral to carry effects on of a properMaraviroc clinical were investigation, investigated on but U87.CD4 its identification cells expressing spurred wild researchers type or towardchimeric the developmentCCR5 and CXCR ofFigure more4. Furthermore, 3. effective Chemical dual-tropic structures a direct influence of CCR5/CXCR4 Maraviroc of the (1 ),drug Plerixafor antagonists was observed (2), and [22]. AMD3451 on the onset (3). of resistance in infected patients [24]. Phase II clinical studies confirmed the susceptibility to Maraviroc of dual About ten years later it was proven that Maraviroc, initially identified as a selective CCR5 inhibitor, mixedAbout tropic ten viruses, years with later an it activity was proven even higher that Maraviroc than that, recor initiallyded identifiedtowards pure as a R5 selective strains [25] CCR5. was also an effective therapeutic alternative against dual mixed tropic HIV strains [23]. Antiviral inhibitor,Nevertheless, was Maraviroc also an effective is indicated therapeutic in combination alternative with againstother antiretroviral dual mixed agents tropic for HIV the strains treatment [23] . effects of Maraviroc were investigated on U87.CD4 cells expressing wild type or chimeric CCR5 Antiviralof only CCR5 effects-tropic of Maraviroc HIV-1 infection, were investigated but not used on for U87.CD4 dual mixed cells viruses expressing in the wildclinic type [26]. or chimeric and CXCR4. Furthermore, a direct influence of the drug was observed on the onset of resistance CCR5A andround CXCR the 4.same Furthermore, time, Hubi an direct and coworkers influence ofhypothesized the drug was that observed the simultaneous on the onset inhibition of resistance of in infected patients [24]. Phase II clinical studies confirmed the susceptibility to Maraviroc of dual inCCR5 infected and CXCR4 patients by [24] transition. Phase metalII clinical complexes studies based confirmed on a tetraazamacrocycle the susceptibility could to Maraviroc be particularly of dual mixed tropic viruses, with an activity even higher than that recorded towards pure R5 strains [25]. mixedeffective tropic for the viruses, treatment with of an HIV activity infection even (Figure higher 4 than) [27,28] that. Thermodynamicrecorded towards properties pure R5 ofstrains bridged [25] . Nevertheless, Maraviroc is indicated in combination with other antiretroviral agents for the treatment Nevertheless,and unbridged Maraviroc metal complexants is indicated were in combination also compared with [29] other. In a antiretroviral successive report, agents the for same the treatmentauthors of only CCR5-tropic HIV-1 infection, but not used for dual mixed viruses in the clinic [26]. ofdeveloped only CCR5 alternative-tropic HIV synthetic-1 infection, routes but to include not used dichloropyridine for dual mixed moietiesviruses in into the the clinic scaffold [26]. of these Around the same time, Hubin and coworkers hypothesized that the simultaneous inhibition of complexantsAround [28]the .same time, Hubin and coworkers hypothesized that the simultaneous inhibition of CCR5 and CXCR4 by transition metal complexes based on a tetraazamacrocycle could be particularly CCR5 and CXCR4 by transition metal complexes based on a tetraazamacrocycle could be particularly effective for the treatment of HIV infection (Figure4)[ 27,28]. Thermodynamic properties of bridged effective for the treatment of HIV infection (Figure 4) [27,28]. Thermodynamic properties of bridged and unbridged metal complexants were also compared [29]. In a successive report, the same authors and unbridged metal complexants were also compared [29]. In a successive report, the same authors developed alternative synthetic routes to include dichloropyridine moieties into the scaffold of these developed alternative synthetic routes to include dichloropyridine moieties into the scaffold of these complexants [28]. complexants [28].

Figure 4. Example of unbridged (4) and cross-bridged (5) tetraazamacrocycles.

Figure 4. Example of unbridged (4) and cross-bridged (5) tetraazamacrocycles. Figure 4. Example of unbridged (4) and cross-bridged (5) tetraazamacrocycles. 2.2. Peptide-Based Antagonists

In a study focusing on the identification of HIV entry inhibitors, peptide triazoles acting as dual antagonists targeting the interaction of gp120 with CD4 and co-receptors CCR5 and CXCR4 were identified. Eleven out of nineteen gp120 alanine mutants were screened by -linked immunosorbent assay (ELISA), leading to compound KR21 as the most active agent in direct binding and competition experiments (Figure5, compound 6). This peptide corresponds to a conserved region of gp120 overlapping with the CD4 binding site. Amino-acid brought to this compound led to a reduction or even complete loss of inhibitory activity. The overall results of this study suggest that KR21 could represent the starting point for the rationale design of smaller dual antagonists [30]. Molecules 2019, 24, x 5 of 11

2.2. Peptide-Based Antagonists In a study focusing on the identification of HIV entry inhibitors, peptide triazoles acting as dual antagonists targeting the interaction of gp120 with CD4 and co-receptors CCR5 and CXCR4 were identified. Eleven out of nineteen gp120 alanine mutants were screened by enzyme-linked immunosorbent assay (ELISA), leading to compound KR21 as the most active agent in direct binding and competition experiments (Figure 5, compound 6). This peptide corresponds to a conserved region of gp120 overlapping with the CD4 binding site. Amino-acid mutation brought to this compound led to a reduction or even complete loss of inhibitory activity. The overall results of this Molecules 2019, 24, 550 5 of 11 study suggest that KR21 could represent the starting point for the rationale design of smaller dual antagonists [30].

FigureFigure 5.5. Chemical structure of KR21 ( (66).). 2.3. Diterpene Derivatives 2.3. Diterpene Derivatives A new approach to the identification of dual inhibitors was proposed by Gama and coworkers, A new approach to the identification of dual inhibitors was proposed by Gama and coworkers, whowho described described aa naturalnatural occurringoccurring ingenolingenol derivative,derivative, a diterpene isolated isolated from from the the tropical tropical shrub shrub EuphorbiaEuphorbia tirucalli tirucalli(Figure (Figure6 ,6 compound, compound 77)[) [31]31].. This compound was further further chemically chemically manipulated manipulated toto give give activeactive cynnamic,cynnamic, caproic, caproic, andand lauric lauric estersesters (Figure 66,, compounds compounds 88––1010)),, which which showed showed promisingpromising antiviralantiviral activity,activity, likelylikely duedue toto thethe downregulation of of membrane membrane receptors receptors CD4, CD4, CCR5 CCR5,, and CXCR4. The compounds were tested against dual tropic HIV-1 strains and the EC values and CXCR4. The compounds were tested against dual tropic HIV-1 strains and the EC50 values50 were werefound found to be to comparable be comparable to those to those of of com commonlymonly used used antiretroviral antiretroviral drugs drugs.. Moreover, Moreover, these these new new derivatives,derivatives, inin particularparticular compoundcompound 9, provedproved to be less toxic than than previously previously discover discovereded ingen ingenolol analogs,analogs, actingacting byby thethe modulationmodulation ofof specificspecific protein kinase C C isoforms involved involved in in the the membrane membrane receptorMoleculesreceptor 201 down-regulation down9, 24, -xregulation [ [32]32].. 6 of 11

Figure 6. Structure of ingenol 7 and its ester derivatives 8–10. Figure 6. Structure of ingenol 7 and its ester derivatives 8–10. 2.4. Pyrazole-Based Antagonists 2.4. Pyrazole-Based Antagonists A composite computational study, based on both virtual screening and statistical approach, led to a seriesA composite of polyheterocyclic computational derivatives study, based active on onboth both virtual CCR5 screening and CXCR4. and statistical The core approach structure, led was to a series of polyheterocyclic derivatives active on both CCR5 and CXCR4. The core structure was represented by a pyrazolo-piperidine nucleus. The most active derivative (Figure 7, compound 11), bearing a benzyl group appended to the pyrazole and a 4-pyridinemethyl linked to the piperidine, showed an IC50 value of 3.8 µM against a CCR5-utilizing HIV-1 strain and an IC50 value of 0.8 µM against a CXCR4-utilizing HIV-1 strain, in MAGI assay. This last consists of a high sensitive competitive in vitro HIV replication method for quantifying viral infectivity. Whereas the benzyl substituent seems necessary for retaining activity, different bonding mode were tolerated for the pyridine ring (Figure 7, compounds 12 and 13). These compounds showed an IC50 value of 17 and 25 µM in the case of the 3-pyridinemethyl derivative and 16 and 5.8 µM in the case of the 2- pyridinemethyl analog against a CCR5- and CXCR4-utilizing HIV-1 strain, respectively. Compound 11 showed also to be active in an assay on Ca2+ flux GPCR signaling, therefore allosterically modulating CXCR4. Furthermore, compound 11 showed to be active against HIV-1 reverse- transcriptase enzyme with an IC50 value of 9.0 μM. Moreover, this compound did not result toxic in the same MAGI assay, at a concentration as high as 300 µM. All these data suggest that this lead compound is warranted for further development for the identification of more active dual chemokine receptor inhibitors [33].

Figure 7. Chemical structure of pyrazolo-piperidine derivatives 11–13.

In a successive computational study, the dynamics of the binding between 11 and both CCR5 and CXCR4 were in depth investigated [34]. The three aromatic rings involved in π-stacking and a positively charged hydrogen bond donor of a piperidine ring were demonstrated to be the main responsible features for the interaction. The replacement of the piperidine ring with a piperazine,

Molecules 2019, 24, x 6 of 11

Figure 6. Structure of ingenol 7 and its ester derivatives 8–10.

2.4. Pyrazole-Based Antagonists Molecules 2019, 24, 550 6 of 11 A composite computational study, based on both virtual screening and statistical approach, led to a series of polyheterocyclic derivatives active on both CCR5 and CXCR4. The core structure was represented by a pyrazolo-piperidinepyrazolo-piperidine nucleus. The most active d derivativeerivative (Figure 77,, compoundcompound 11), bearing a benzyl group appended to the pyrazole and a 4-pyridinemethyl4-pyridinemethyl linked to the piperidine,piperidine, showed an IC50 valuevalue of 3.8 µMµM against a CCR5-utilizingCCR5-utilizing HIV-1HIV-1 strainstrain andand anan ICIC5050 value of 0.8 µµMM against aa CXCR4-utilizing CXCR4-utilizing HIV-1 HIV strain,-1 strain, in MAGI in MAGI assay. assayThis last. This consists last of consist a highs sensitiveof a high competitive sensitive incompetitive vitro HIV replicationin vitro HIV method replication for quantifying method for viral quantifying infectivity. viral Whereas infectivity. the benzyl Whereas substituent the benzyl seems necessarysubstituent for seems retaining necessary activity, for different retaining bonding activity, mode different were tolerated bonding for mode the pyridine were tolerat ringed (Figure for the7, compoundspyridine ring12 (Figureand 13 7)., compound These compoundss 12 and showed13). These an compounds IC50 value ofshowed 17 and an 25 ICµ50M value in the of case 17 and of the 25 3-pyridinemethylµM in the case of derivative the 3-pyridinemethyl and 16 and 5.8 derivativeµM in the andcase of16 the and 2-pyridinemethyl 5.8 µM in the analogcase of againstthe 2- apyridinemethyl CCR5- and CXCR4-utilizing analog against a HIV-1 CCR5 strain,- and CXCR4 respectively.-utilizing Compound HIV-1 strai11n,showed respectively. also toCompound be active in11 anshowed assay on also Ca 2+ to flux be active GPCR in signaling, an assay therefore on Ca2+ allosterically flux GPCR modulating signaling, therefore CXCR4. Furthermore,allosterically compoundmodulating11 CXCR4.showed Furthermore, to be active against compound HIV-1 reverse-transcriptase11 showed to be active enzyme against with an HIV IC-501 value reverse of- 9.0transcriptaseµM. Moreover, enzyme this with compound an IC50 didvalue not of result 9.0 μM. toxic Moreover, in the same this MAGIcompound assay, di atd anot concentration result toxic asin highthe same as 300 MAGIµM.All assay, these at data a concentration suggest that thisas high lead as compound 300 µM. isAll warranted these data for suggest further that development this lead forcompound the identification is warrant ofed more for further active development dual chemokine for receptorthe identification inhibitors of [ 33more]. active dual chemokine receptor inhibitors [33].

Figure 7. Chemical structure of pyrazolo-piperidinepyrazolo-piperidine derivatives 11–1313..

In aa successivesuccessive computationalcomputational study, study the, the dynamics dynamics of theof the binding binding between between11 and 11 and both bot CCR5h CCR5 and CXCR4and CXCR4 were were in depth in depth investigated investigated [34]. The [34] three. The aromatic three aromatic rings involved rings involved in π-stacking in π- andstacking a positively and a chargedpositively hydrogen charged bond hydrogen donor bond of a donorpiperidine of a ringpiperidine were demonstrated ring were demonstrated to be the main to be responsible the main Molecules 2019, 24, x 7 of 11 featuresresponsible for thefeatures interaction. for the Theinteraction. replacement The replacement of the piperidine of the ring piperidine with a piperazine,ring with a leadingpiperazine, to a double protonated species interacting with the negatively charged glutamates and aspartates within the leading to a double protonated species interacting with the negatively charged glutamates and active site, was planned in order to strengthen the protein-ligand interaction. Accordingly, compound aspartates within the active site, was planned in order to strengthen the protein-ligand interaction. 14 (Figure8) was synthesized and demonstrated to have a more favorable interaction compared to Accordingly, compound 14 (Figure 8) was synthesized and demonstrated to have a more favorable 11, after being docked into the active site of both co-receptors [34]. These results suggested that interaction compared to 11, after being docked into the active site of both co-receptors [34]. These further insights into the molecular dynamics of such compounds and CCR5/CXCR4 could lead to the results suggested that further insights into the molecular dynamics of such compounds and identification of more effective dual antagonists. CCR5/CXCR4 could lead to the identification of more effective dual antagonists.

Figure 8. Chemical structure of piperidine derivative 14. Figure 8. Chemical structure of piperidine derivative 14. 2.5. The Suramin Analog NF279 2.5. The Suramin Analog NF279 Inhibition by selective antagonists of P2X1R, a receptor involved in the HIV-1 fusion and replication,Inhibition could by represent selective antagonists an alternative of strategy P2X1R, a to receptor contrast involved the viral in infection. the HIV Compound-1 fusion and15, alsoreplication, known could as NF279 represent (Figure an9), alternative an analog ofstrategy the anti-parasite to contrast drug the viral suramin, infection. was initiallyCompound found 15 to, also be asknown a selective as NF279 P2X1 (Figure receptor 9), antagonist, an analog andof the showed anti-parasite a noteworthy drug suramin, antiviral activitywas initially [35]. Furtherfound to studies be as a selective P2X1 , and showed a noteworthy antiviral activity [35]. Further studies proved that HIV-1 entry inhibitory activity of compound 15 was not related to a direct interaction with P2X1R, but rather to the capability of this compound to act as dual CCR5/CXCR4 co-receptor antagonist. This feature was assessed by the suppression of cellular calcium responses CCR5/CXCR4 mediated. Thus, NF279 could represent the lead of a new class of dual-coreceptor inhibitors [36].

Figure 9. Chemical structure of compound 15.

2.6. The Cumarin-Based Ligand GUT-70 It is known that several derivatives of the natural product coumarin are endowed with several pharmacological properties, including anti-viral activity [37]. Compound 16, a tricyclic coumarin also known as GUT-70 (Figure 10), was demonstrated to be able to reduce cell membrane fluidity by a fluorescent depolarization study conducted on MOLT-4 and PM1-CCR5 T cell lines. This property would suggest its potential use against the HIV-1 entry. Furthermore, GUT-70 is capable of down- regulating the expression of CD4, CCR5, and CXCR4 receptors on the cell surface in a dose-dependent manner, therefore representing a starting point for the development of novel tools against HIV-1 infection [38]. However, this compound showed an unfavorable toxicity profile, due to a noteworthy cytotoxicity [39].

Molecules 2019, 24, x 7 of 11 leading to a double protonated species interacting with the negatively charged glutamates and aspartates within the active site, was planned in order to strengthen the protein-ligand interaction. Accordingly, compound 14 (Figure 8) was synthesized and demonstrated to have a more favorable interaction compared to 11, after being docked into the active site of both co-receptors [34]. These results suggested that further insights into the molecular dynamics of such compounds and CCR5/CXCR4 could lead to the identification of more effective dual antagonists.

Figure 8. Chemical structure of piperidine derivative 14.

2.5. The Suramin Analog NF279 Inhibition by selective antagonists of P2X1R, a receptor involved in the HIV-1 fusion and replication,Molecules 2019 ,could24, 550 represent an alternative strategy to contrast the viral infection. Compound 157, also of 11 known as NF279 (Figure 9), an analog of the anti-parasite drug suramin, was initially found to be as a selective P2X1 receptor antagonist, and showed a noteworthy antiviral activity [35]. Further studies proved that HIV-1 entry inhibitory activity of compound 15 was not related to a direct interaction proved that HIV-1 entry inhibitory activity of compound 15 was not related to a direct interaction with P2X1R, but rather to the capability of this compound to act as dual CCR5/CXCR4 co-receptor with P2X1R, but rather to the capability of this compound to act as dual CCR5/CXCR4 co-receptor antagonist. This feature was assessed by the suppression of cellular calcium responses CCR5/CXCR4 antagonist. This feature was assessed by the suppression of cellular calcium responses CCR5/CXCR4 mediated. Thus, NF279 could represent the lead of a new class of dual-coreceptor inhibitors [36]. mediated. Thus, NF279 could represent the lead of a new class of dual-coreceptor inhibitors [36].

Figure 9. Chemical structure of compound 15. Figure 9. Chemical structure of compound 15. 2.6. The Cumarin-Based Ligand GUT-70 2.6. The Cumarin-Based Ligand GUT-70 It is known that several derivatives of the natural product coumarin are endowed with several pharmacologicalIt is known that properties, severalincluding derivatives anti-viral of the natural activity product [37]. Compound coumarin16 are, a endowed tricyclic coumarin with several also pharmacologicalknown as GUT-70 properties, (Figure 10 including), was demonstrated anti-viral activity to be [37] able. Compound to reduce cell 16, membranea tricyclic coumarin fluidity byalso a knownfluorescent as GUT depolarization-70 (Figure study 10), was conducted demonstrated on MOLT-4 to be and able PM1-CCR5 to reduce T cell cell lines. membrane This property fluidity would by a fluorescentsuggest its potentialdepolarization use against study the conducted HIV-1 entry. on MOLT Furthermore,-4 and PM1 GUT-70-CCR5 is capable T cell lines. of down-regulating This property wouldthe expression suggest ofits CD4, potential CCR5, use and against CXCR4 the receptors HIV-1 entry. on the Furthermore, cell surface inGUT a dose-dependent-70 is capable of manner, down- regulatingtherefore representing the expression a starting of CD4, point CCR5 for, and the CXCR4 development receptors of novelon the tools cell surface against in HIV-1 a dose infection-dependent [38]. Molecules 2019, 24, x 8 of 11 manner,However, therefore this compound representing showed a anstarting unfavorable point toxicityfor the profile,development due to aof noteworthy novel tools cytotoxicity against HIV [39-1]. infection [38]. However, this compound showed an unfavorable toxicity profile, due to a noteworthy cytotoxicity [39].

Figure 10. Chemical structure of compound 16. Figure 10. Chemical structure of compound 16. 2.7. Penicillixanthone A 2.7. PenicillixanthoneIt is well known A that HIV fusion can be humped by a peptide portion of (HR2 domain). An antiviralIt is well effect known has that been HIV demonstrated fusion can be when humped this peptide by a peptide portion portion was conjugated of gp41 (HR2 to cell domain). membrane An antiviralproteins. effect In particular, has been a 34demonstrated amino acid peptide when this from peptide HR2 (C34, portion corresponding was conjugated to amino to acidscell membrane 628–661 in proteins.HxB2) [40 In,41 particular,] conjugated a to34 theamino amino acid termini peptide of CCR5from HR2 or CXCR4 (C34, exhibitedcorresponding potent to and amino broad acids inhibition 628– 661against in HxB2) several [40,41 HIV-1] conjugated strains [42 to]. the Potent amino anti-HIV-1 termini of activity CCR5 or was CXCR4 showed exhibited by penicillixanthone potent and broad A inhibition(PXA, Figure against 11, compound several17 HIV), a natural-1 strains xanthone [42]. dimer Potent isolated anti- fromHIV- the1 activityfungus Aspergillus was showed fumigatus by . pTheenicillixanthone actual mechanism A (PXA, of action Figure of PXA 11, compound was inferred 17 by), amolecular natural xanthone modeling dimerstudies, isolated which suggested from the fungusa putative Aspergillus interaction fumigat bindingus. mode The actual with both mechanism CCR5 and of CXCR4 action ofreceptors. PXA was The inferred IC50 values by molecular calculated modelingby in vitro studies,studies which were 0.36 suggested and 0.26 a µputativeM against interaction CCR5- and binding CXCR4-tropic mode with HIV-1 both strains, CCR5 and respectively. CXCR4 receptors.PXA showed The only IC50 a values moderate calculated cytotoxicity by in evaluated vitro studies by a colorimetricwere 0.36 and XTT 0.26 assay μM against against TZM-bl CCR5- cells,and CXCR4with an-tropic IC50 of HIV 20.6-1µ strains,M. This respectively. compound couldPXA showed then represent only a moderate a lead for cytotoxicity the development evaluated of HIV-1 by a colorimetricdual co-receptor XTT antagonistsassay against [43 ].TZM-bl cells, with an IC50 of 20.6 μM. This compound could then represent a lead for the development of HIV-1 dual co-receptor antagonists [43].

Figure 11. Chemical structure of compound 17.

Considering all the above remarks, the identification of dual CCR5/CXCR4 co-receptor antagonists is still an attractive field of anti-HIV research. In fact, the progression of HIV infection is associated with the skill of the virus to switch from one co-receptor to another. Although our understanding of the viral resistance mechanism is not yet complete, the alternative therapeutic option based on the inhibition of membrane co-receptors sounds still appealing. Such an approach could gain particular importance in the case of anti-HIV treatment failure with classic HAART therapy. After the identification of effective antagonists, the next step could reside in the choice of a specific therapy based on the use of selective or dual agents, or even a combination of both types.

3. Conclusion Successful applications of ligand-based models and recent insights on the mechanism of HIV replication prompted the research of a new strategy aimed at preventing viral adhesion and spread. A promising approach is based on the employment of agents able to antagonize the interaction of viral proteins with the host cell membrane receptor CD4 and co-receptors CCR5 and CXCR4. The

Molecules 2019, 24, x 8 of 11

Figure 10. Chemical structure of compound 16.

2.7. Penicillixanthone A It is well known that HIV fusion can be humped by a peptide portion of gp41 (HR2 domain). An antiviral effect has been demonstrated when this peptide portion was conjugated to cell membrane proteins. In particular, a 34 amino acid peptide from HR2 (C34, corresponding to amino acids 628– 661 in HxB2) [40,41] conjugated to the amino termini of CCR5 or CXCR4 exhibited potent and broad inhibition against several HIV-1 strains [42]. Potent anti-HIV-1 activity was showed by penicillixanthone A (PXA, Figure 11, compound 17), a natural xanthone dimer isolated from the fungus Aspergillus fumigatus. The actual mechanism of action of PXA was inferred by molecular modeling studies, which suggested a putative interaction binding mode with both CCR5 and CXCR4 receptors. The IC50 values calculated by in vitro studies were 0.36 and 0.26 μM against CCR5- and CXCR4-tropic HIV-1 strains, respectively. PXA showed only a moderate cytotoxicity evaluated by a Molecules 2019, 24, 550 8 of 11 colorimetric XTT assay against TZM-bl cells, with an IC50 of 20.6 μM. This compound could then represent a lead for the development of HIV-1 dual co-receptor antagonists [43].

Figure 11. 17 Figure 11. Chemical structure of compound 17. Considering all the above remarks, the identification of dual CCR5/CXCR4 co-receptor antagonists Considering all the above remarks, the identification of dual CCR5/CXCR4 co-receptor is still an attractive field of anti-HIV research. In fact, the progression of HIV infection is associated with antagonists is still an attractive field of anti-HIV research. In fact, the progression of HIV infection is the skill of the virus to switch from one co-receptor to another. Although our understanding of the viral associated with the skill of the virus to switch from one co-receptor to another. Although our resistance mechanism is not yet complete, the alternative therapeutic option based on the inhibition of understanding of the viral resistance mechanism is not yet complete, the alternative therapeutic membrane co-receptors sounds still appealing. Such an approach could gain particular importance in option based on the inhibition of membrane co-receptors sounds still appealing. Such an approach the case of anti-HIV treatment failure with classic HAART therapy. After the identification of effective could gain particular importance in the case of anti-HIV treatment failure with classic HAART antagonists, the next step could reside in the choice of a specific therapy based on the use of selective or therapy. After the identification of effective antagonists, the next step could reside in the choice of a dual agents, or even a combination of both types. specific therapy based on the use of selective or dual agents, or even a combination of both types. 3. Conclusions 3. Conclusion Successful applications of ligand-based models and recent insights on the mechanism of HIV Successful applications of ligand-based models and recent insights on the mechanism of HIV replication prompted the research of a new strategy aimed at preventing viral adhesion and spread. replication prompted the research of a new strategy aimed at preventing viral adhesion and spread. A promising approach is based on the employment of agents able to antagonize the interaction of viral A promising approach is based on the employment of agents able to antagonize the interaction of proteins with the host cell membrane receptor CD4 and co-receptors CCR5 and CXCR4. The viral viral proteins with the host cell membrane receptor CD4 and co-receptors CCR5 and CXCR4. The tropism is in fact thoroughly influenced by the expression of one or both co-receptors. The identification of selective co-receptor antagonists could be particularly effective in preventing viral infection. However, the capability of the virus to switch tropism could reduce drug effectiveness. In this context, the discovery of dual CCR5 and CXCR4 could lead to even more appealing anti-viral agents, able to prevent the replication of the more virulent dual-tropic HIV strains. The studies so far conducted for the identification of dual inhibitors have led to interesting candidates to be further developed, even if several of the identified compounds suffer from an inadequate safety and profile. Most of the analyzed papers deal with in vitro and in silico evaluations, which often do not reflect therapeutic potentials for a suitable clinical application. Rationale multidisciplinary efforts combining computational and biological techniques are then required for the design of leads to be developed toward clinically useful potent antiviral agents.

Author Contributions: Conceptualization: F.G., B.R.; Supervision: S.A., A.G.; Writing—original draft preparation: M.A.O., P.T., G.I., F.G.; Writing—review and editing: V.S., M.D.L., F.G., B.R. All authors agree with the final version of the manuscript. The authors declare that the content of this paper has not been published or submitted for publication elsewhere. Funding: This study was partly funded by PRIN (Progetti di Rilevante Interesse Nazionale) Grant 2015W729WH_007 from the MIUR, Italy. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Cobucci, R.N.; Lima, P.H.; de Souza, P.C.; Costa, V.V.; Cornetta Mda, C.; Fernandes, J.V.; Goncalves, A.K. Assessing the impact of HAART on the incidence of defining and non-defining AIDS among patients with HIV/AIDS: A systematic review. J. Infect. Public Health 2015, 8, 1–10. [CrossRef][PubMed] 2. Oliva-Moreno, J.; Trapero-Bertran, M. Economic Impact of HIV in the Highly Active Antiretroviral Therapy Era—Reflections Looking Forward. AIDS Rev. 2018, 20, 226–235. [CrossRef][PubMed] Molecules 2019, 24, 550 9 of 11

3. Shepherd, A.J.; Loo, L.; Mohapatra, D.P. Chemokine co-receptor CCR5/CXCR4-dependent modulation of Kv2.1 channel confers acute neuroprotection to HIV-1 glycoprotein gp120 exposure. PLoS ONE 2013, 8, e76698. [CrossRef][PubMed] 4. Dragic, T.; Litwin, V.; Allaway, G.P.; Martin, S.R.; Huang, Y.; Nagashima, K.A.; Cayanan, C.; Maddon, P.J.; Koup, R.A.; Moore, J.P.; et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 1996, 381, 667–673. [CrossRef][PubMed] 5. Scarlatti, G.; Tresoldi, E.; Bjorndal, A.; Fredriksson, R.; Colognesi, C.; Deng, H.K.; Malnati, M.S.; Plebani, A.; Siccardi, A.G.; Littman, D.R.; et al. In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression. Nat. Med. 1997, 3, 1259–1265. [CrossRef][PubMed] 6. Grande, F.; Garofalo, A.; Neamati, N. Small molecules anti-HIV therapeutics targeting CXCR4. Curr. Pharm. Des. 2008, 14, 385–404. [PubMed] 7. Singh, I.P.; Chauthe, S.K. Small molecule HIV entry inhibitors: Part II. Attachment and fusion inhibitors: 2004-2010. Expert Opin. Ther. Pat. 2011, 21, 399–416. [CrossRef] 8. Oppermann, M. Chemokine receptor CCR5: Insights into structure, function, and regulation. Cell Signal. 2004, 16, 1201–1210. [CrossRef] 9. Peng, P.; Chen, H.; Zhu, Y.; Wang, Z.; Li, J.; Luo, R.H.; Wang, J.; Chen, L.; Yang, L.M.; Jiang, H.; et al. Structure-Based Design of 1-Heteroaryl-1,3-propanediamine Derivatives as a Novel Series of CC-Chemokine Receptor 5 Antagonists. J. Med. Chem. 2018, 61, 9621–9636. [CrossRef] 10. Wu, B.; Chien, E.Y.; Mol, C.D.; Fenalti, G.; Liu, W.; Katritch, V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F.C.; et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 2010, 330, 1066–1071. [CrossRef] 11. Chien, H.C.; Chan, P.C.; Tu, C.C.; Day, Y.J.; Hung, L.M.; Juan, C.C.; Tian, Y.F.; Hsieh, P.S. Importance of PLC-Dependent PI3K/AKT and AMPK Signaling in RANTES/CCR5 Mediated Macrophage Chemotaxis. Chin. J. Physiol. 2018, 266–279. [CrossRef][PubMed] 12. Smith, N.; Pietrancosta, N.; Davidson, S.; Dutrieux, J.; Chauveau, L.; Cutolo, P.; Dy, M.; Scott-Algara, D.; Manoury, B.; Zirafi, O.; et al. Natural amines inhibit activation of human plasmacytoid dendritic cells through CXCR4 engagement. Nat. Commun. 2017, 8, 14253. [CrossRef][PubMed] 13. Rangel, H.R.; Bello, G.; Villalba, J.A.; Sulbaran, Y.F.; Garzaro, D.; Maes, M.; Loureiro, C.L.; de Waard, J.H.; Pujol, F.H. The Evolving HIV-1 Epidemic in Warao Amerindians Is Dominated by an Extremely High Frequency of CXCR4-Utilizing Strains. AIDS Res. Hum. Retrovir. 2015, 31, 1265–1268. [CrossRef][PubMed] 14. Gupta, S.; Prosser, A.R.; Cox, B.D.; Wilson, L.J.; Liotta, D. Design and synthesis of dual-tropic HIV entry inhibitors that utilize a homologous CCR5/CXCR4 binding site. In Proceedings of the 249th ACS National Meeting & Exposition, Denver, CO, USA, 22–26 March 2015. 15. Gupta, S.; Prosser, A.R.; Cox, B.D.; Wilson, L.J.; Liotta, D.C. Design and synthesis of Dual Tropic HIV entry inhibitors that utilize a homologous CCR5/CXCR4 binding site. In Proceedings of the 66th Southeast Regional Meeting of the American Chemical Society, Nashville, TN, USA, 16–19 October 2014. 16. Woollard, S.M.; Kanmogne, G.D. Maraviroc: A review of its use in HIV infection and beyond. Drug Des. Dev. Ther. 2015, 9, 5447–5468. 17. Grande, F.; Giancotti, G.; Ioele, G.; Occhiuzzi, M.A.; Garofalo, A. An update on small molecules targeting CXCR4 as starting points for the development of anti-cancer therapeutics. Eur. J. Med. Chem. 2017, 139, 519–530. [CrossRef][PubMed] 18. Schols, D.; Struyf, S.; Van Damme, J.; Este, J.A.; Henson, G.; De Clercq, E. Inhibition of T-tropic HIV strains by selective antagonization of the chemokine receptor CXCR4. J. Exp. Med. 1997, 186, 1383–1388. [CrossRef] [PubMed] 19. Aquaro, S.; Perno, C.F.; Balestra, E.; Balzarini, J.; Cenci, A.; Francesconi, M.; Panti, S.; Serra, F.; Villani, N.; Calio, R. Inhibition of replication of HIV in primary monocyte/macrophages by different antiviral drugs and comparative efficacy in lymphocytes. J. Leukoc. Biol. 1997, 62, 138–143. [CrossRef][PubMed] 20. Donzella, G.A.; Schols, D.; Lin, S.W.; Este, J.A.; Nagashima, K.A.; Maddon, P.J.; Allaway, G.P.; Sakmar, T.P.; Henson, G.; De Clercq, E.; et al. AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor. Nat. Med. 1998, 4, 72–77. [CrossRef][PubMed] 21. Princen, K.; Hatse, S.; Vermeire, K.; Aquaro, S.; De Clercq, E.; Gerlach, L.O.; Rosenkilde, M.; Schwartz, T.W.; Skerlj, R.; Bridger, G.; et al. Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist. J. Virol. 2004, 78, 12996–13006. [CrossRef][PubMed] Molecules 2019, 24, 550 10 of 11

22. Kim, M.B.; Giesler, K.E.; Tahirovic, Y.A.; Truax, V.M.; Liotta, D.C.; Wilson, L.J. CCR5 receptor antagonists in preclinical to phase II clinical development for treatment of HIV. Expert Opin. Investig. Drugs 2016, 25, 1377–1392. [CrossRef][PubMed] 23. Surdo, M.; Balestra, E.; Saccomandi, P.; Di Santo, F.; Montano, M.; Di Carlo, D.; Sarmati, L.; Aquaro, S.; Andreoni, M.; Svicher, V.; et al. Inhibition of dual/mixed tropic HIV-1 isolates by CCR5-inhibitors in primary lymphocytes and macrophages. PLoS ONE 2013, 8, e68076. [CrossRef][PubMed] 24. Cavarelli, M.; Mainetti, L.; Pignataro, A.R.; Bigoloni, A.; Tolazzi, M.; Galli, A.; Nozza, S.; Castagna, A.; Sampaolo, M.; Boeri, E.; et al. Complexity and dynamics of HIV-1 chemokine receptor usage in a multidrug- resistant adolescent. AIDS Res. Hum. Retrovir. 2014, 30, 1243–1250. [CrossRef][PubMed] 25. Surdo, M.; Alteri, C.; Puertas, M.C.; Saccomandi, P.; Parrotta, L.; Swenson, L.; Chapman, D.; Costa, G.; Artese, A.; Balestra, E.; et al. Effect of maraviroc on non-R5 tropic HIV-1: Refined analysis of subjects from the phase IIb study A4001029. Clin. Microbiol. Infect. 2015, 21, 103e1–103e6. [CrossRef][PubMed] 26. Serrano-Villar, S.; Caruana, G.; Zlotnik, A.; Perez-Molina, J.A.; Moreno, S. Effects of Maraviroc versus in Combination with Zidovudine- on the CD4/CD8 Ratio in Treatment-Naive HIV-Infected Individuals. Antimicrob. Agents Chemother. 2017, 61, e01763-17. [CrossRef] [PubMed] 27. Hubin, T.J.; Archibald, S.J.; Won, P.; Birdsong, O.C.; Epley, B.M.; Klassen, S.L.; Schols, D. Synthesis and evaluation of transition metal complex dual CXCR4/CCR5 antagonists. In Proceedings of the 245th ACS National Meeting & Exposition, New Orleans, LA, USA, 7–11 April 2013. 28. Davilla, D.; Birdsong, O.; Schols, D.; Archibald, S.; Hubin, T. Bis- and pendant armed tetraazamacrocycle transition metal complex dual CXCR4/CCR5 antagonists. In Proceedings of the 251st ACS National Meeting & Exposition, San Diego, CA, USA, 13–17 March 2016. 29. Available online: https://www.swosu.edu/academics/jur/docs/transitionmetals-v1.pdf (accessed on 31 January 2019). 30. Tuzer, F.; Madani, N.; Kamanna, K.; Zentner, I.; LaLonde, J.; Holmes, A.; Upton, E.; Rajagopal, S.; McFadden, K.; Contarino, M.; et al. HIV-1 Env gp120 structural determinants for peptide triazole dual receptor site antagonism. Proteins 2013, 81, 271–290. [CrossRef][PubMed] 31. Abreu, C.M.; Price, S.L.; Shirk, E.N.; Cunha, R.D.; Pianowski, L.F.; Clements, J.E.; Tanuri, A.; Gama, L. Dual role of novel ingenol derivatives from Euphorbia tirucalli in HIV replication: Inhibition of de novo infection and activation of viral LTR. PLoS ONE 2014, 9, e97257. [CrossRef] 32. Spina, C.A.; Anderson, J.; Archin, N.M.; Bosque, A.; Chan, J.; Famiglietti, M.; Greene, W.C.; Kashuba, A.; Lewin, S.R.; Margolis, D.M.; et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog. 2013, 9, e1003834. [CrossRef] [PubMed] 33. Cox, B.D.; Prosser, A.R.; Sun, Y.; Li, Z.; Lee, S.; Huang, M.B.; Bond, V.C.; Snyder, J.P.; Krystal, M.; Wilson, L.J.; et al. Pyrazolo-Piperidines Exhibit Dual Inhibition of CCR5/CXCR4 HIV Entry and Reverse Transcriptase. ACS Med. Chem. Lett. 2015, 6, 753–757. [CrossRef] 34. Taylor, C.A.; Miller, B.R., 3rd; Parish, C.A. Design and computational support for the binding stability of a new CCR5/CXCR4 dual tropic inhibitor: Computational design of a CCR5/CXCR4 drug. J. Mol. Graph. Model. 2017, 75, 71–79. [CrossRef] 35. Marin, M.; Du, Y.; Giroud, C.; Kim, J.H.; Qui, M.; Fu, H.; Melikyan, G.B. High-Throughput HIV-Cell Fusion Assay for Discovery of Virus Entry Inhibitors. Assay Drug Dev. Technol. 2015, 13, 155–166. [CrossRef] 36. Giroud, C.; Marin, M.; Hammonds, J.; Spearman, P.; Melikyan, G.B. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions. J. Virol. 2015, 89, 9368–9382. [CrossRef][PubMed] 37. Venugopala, K.N.; Rashmi, V.; Odhav, B. Review on natural coumarin lead compounds for their pharmacological activity. BioMed Res. Int. 2013.[CrossRef][PubMed] 38. Matsuda, K.; Hattori, S.; Kariya, R.; Komizu, Y.; Kudo, E.; Goto, H.; Taura, M.; Ueoka, R.; Kimura, S.; Okada, S. Inhibition of HIV-1 entry by the tricyclic coumarin GUT-70 through the modification of membrane fluidity. Biochem. Biophys. Res. Commun. 2015, 457, 288–294. [CrossRef][PubMed] 39. Kudo, E.; Taura, M.; Matsuda, K.; Shimamoto, M.; Kariya, R.; Goto, H.; Hattori, S.; Kimura, S.; Okada, S. Inhibition of HIV-1 replication by a tricyclic coumarin GUT-70 in acutely and chronically infected cells. Bioorg. Med. Chem. Lett. 2013, 23, 606–609. [CrossRef][PubMed] Molecules 2019, 24, 550 11 of 11

40. Tozser, J.; Blaha, I.; Copeland, T.D.; Wondrak, E.M.; Oroszlan, S. Comparison of the HIV-1 and HIV-2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag-Pol polyproteins. FEBS Lett. 1991, 281, 77–80. [CrossRef] 41. Gorelick, R.J.; Henderson, L.E. Human and AIDS 1994-Part III; Elsevier: Amsterdam, The Netherlands, 2017; p. 9. 42. Leslie, G.J.; Wang, J.; Richardson, M.W.; Haggarty, B.S.; Hua, K.L.; Duong, J.; Secreto, A.J.; Jordon, A.P.; Romano, J.; Kumar, K.E.; et al. Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus. PLoS Pathog. 2016, 12, e1005983. [CrossRef] 43. Tan, S.; Yang, B.; Liu, J.; Xun, T.; Liu, Y.; Zhou, X. Penicillixanthone A, a marine-derived dual-coreceptor antagonist as anti-HIV-1 agent. Nat. Prod. Res. 2017, 1–5. [CrossRef]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).