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Polymerase Acidic Protein-Basic Protein 1 (PA-PB1) Protein-protein Interaction as a Target for Next-generation Anti-influenza Therapeutics Serena Massari, Laura Goracci, Jenny Desantis, and Oriana Tabarrini J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01474 • Publication Date (Web): 05 Apr 2016 Downloaded from http://pubs.acs.org on April 6, 2016

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Journal of Medicinal Chemistry

Polymerase Acidic Protein-Basic Protein 1 (PA-PB1) Protein-protein Interaction as a Target for Next-generation Anti-influenza Therapeutics

Serena Massari,§,* Laura Goracci,¥,* Jenny Desantis,§ and Oriana Tabarrini.§

Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, Italy and Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy.

*

To whom correspondence should be addressed. For SM: phone, + 39 075 585 5146; fax, +39 075

585 5115; e-mail, [email protected]. For LG: phone, +39 075 585 5632; fax, +39 075 45646; e-mail, [email protected].

§

Department of Pharmaceutical Sciences.

¥

Department of Chemistry, Biology, and Biotechnology. 1 ACS Paragon Plus Environment


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ABSTRACT The limited therapeutic options against the influenza virus (flu) and increasing challenges in drug resistance make the search for next-generation agents imperative. In this context, heterotrimeric viral PA/PB1/PB2 RNA-dependent-RNA-polymerase is an attractive target for a challenging but strategic protein-protein interaction (PPI) inhibition approach. Since 2012, the inhibition of the polymerase PA-PB1 subunits interface has become an active field of research, following the publication of PA-PB1 crystal structures. In this Perspective, the validity of flu polymerase as a drug target and its inhibition through a PPI inhibition strategy will be briefly discussed, including a comprehensive analysis of available PA-PB1 structures. An overview of all the reported PA-PB1 complex formation inhibitors will be provided; approaches used for identification of the inhibitors, the hit-to-lead studies and the emerged structure-activity relationship will be described. In addition to highlighting the strengths and weaknesses of all the PA-PB1 heterodimerization inhibitors, we will analyze their hypothesized binding modes and alignment with a pharmacophore model we have developed.

1. INTRODUCTION Influenza virus (flu) is a major cause of viral respiratory infections. Although influenza is a selflimiting disease in healthy adults, it is a life-threatening event associated with severe secondary infections for high-risk populations, i.e., the elderly, infants, and the chronically ill. In addition to the host immune response, the course of the disease, ranging from mild to fatal, depend on the flu strain. There are three types of flu, A, B and C, that are classified according to the antigenicity of their nucleoprotein (NP) and matrix protein 1 (M1). FluA strains infect a wide range of avian and mammalian hosts, while fluB infects only humans. FluAs are further divided into subtypes based on the nature of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). FluA and fluB infections usually occur in seasonal epidemics, which result in approximately 3 to 5 million cases of severe illness and approximately 250 000 to 500 000 deaths each year all over the 2 ACS Paragon Plus Environment

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world, as reported by the World Health Organization (WHO).1 The high rates of morbidity and mortality caused by influenza epidemics can dramatically increase during severe pandemic outbreaks, which arise when highly virulent fluA strains acquire the ability to spread easily among human beings. Three documented influenza pandemics occurred in the 20th century. In 2009, the WHO announced the first pandemic of the 21st century that resulted from the new “swine flu” H1N1pdm09 strain,2,3 which has currently joined the repertoire of typical seasonal flu strains circulating within the human population, such as fluA subtype H3N2 and fluBs.4 There is great concern that another unpredictable devastating pandemic is inevitable, perpetuated by the circulation of fluA strains and even more by the continuous emergence of new fluA strains; these strains have not yet acquired the capacity to transmit between people, but they sporadically cross the species barrier and infect humans. The highly pathogenic “avian flu” H5N1 strain, which caused the unprecedented emergence of infections in 1997 and re-emerged in 2003 with a higher case-fatality rate, is now circulating in Asia, Africa, and Europe among poultry and periodically infects humans.5 In 2013, three novel avian fluA subtypes breached the animal-human host species barrier in Asia: fluA H7N9, which caused severe human infections and 49 deaths over a two-month span,6 fluA H6N1, albeit with low pathogenicity,7 and fluA H10N8 that caused three cases of human infections, two of which were fatal.8 Concerns regarding the pandemic threat are heightened by the inadequacy of current prophylactic strategies and treatments for flu infections. Vaccination is the most common prophylaxis, but this must be updated each year to keep up with virus antigenic variations; the effectiveness of vaccines is variable within host populations, and they are ineffective against highly virulent fluA pandemic strains.9 Thus, antivirals are the only weapon against the flu pandemic, especially when vaccines are ineffective or unavailable and in the early stages of a pandemic when lag time is needed to produce a new vaccine. Until now, the antiviral armamentarium to treat flu infections has been limited to a few licensed drugs (Figure 1),10 two M2 ion channel inhibitors, amantadine and rimantadine (adamantanes), and two NA inhibitors, inhaled zanamivir and oral oseltamivir phosphate. Two 3 ACS Paragon Plus Environment


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additional NA inhibitors, intravenous peramivir11 and inhaled laninamivir octanoate,12 and the first polymerase inhibitor, oral favipiravir,13 were recently approved in some Asian countries. However, serious side effects, low efficacy, and the emergence of widespread resistance14-17 have limited the clinical use of licensed anti-flu drugs, to the point where M2 inhibitors are no longer recommended. Currently, the WHO recommends NA inhibitors as the first-line treatment for people who require anti-flu therapy.1 NA inhibitors have an improved safety profile over M2 inhibitors and are active against both fluA and B stains; however, many FluAs are already resistant to NA inhibitors,18-20 and fluBs with a reduced sensitivity to NA inhibitors are emerging,21,22 limiting their range of use.

Figure 1. Structure of licensed anti-flu drugs.

As a member of the Orthomyxoviridae family, fluA possesses a negative-sense single-stranded RNA genome, which is organized into eight different viral RNA (vRNA) segments that encode for 10 identified viral proteins (Figure 2).23,24

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Figure 2. Schematic representation of a fluA virus particle. The fluA virion is constituted by a lipid bilayer envelope that exposes three viral proteins on its surface: the antigenic proteins hemagglutinin (HA) and neuraminidase (NA), and the M2 ion channel. Viral matrix protein M1 coats the inside of the viral membrane. The viral genome lies within the M1 inner layer, which consists of eight negative-sense single-stranded RNA molecules that are encapsidated with nucleoprotein (NP) and associated with the viral polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA) to form the viral ribonucleoprotein (vRNP) complex. Two additional viral proteins encoded by the virus are the non-structural protein 1 (NS1), which is a multifunctional protein and a virulence factor, and the non-structural protein 2 (NS2), also known as the nuclear export protein (NEP) due to its involvement in the nuclear export of viral vRNP.



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Each segment is associated with multiple copies of viral NP and with the viral RNA-dependent RNA polymerase (RdRP) to form the viral ribonucleoprotein (vRNP) complex. The vRNP constitutes the active transcription and replication component unit,25 in which the RdRP performs both the distinct processes of transcription and replication using the same vRNA.26-28 FluA RdRP is a large heterotrimeric complex comprising the polymerase acidic protein (PA), the polymerase basic protein 1 (PB1), and the polymerase basic protein 2 (PB2). The three subunits, of which PB1 is the body of the trimer, interact with each other extensively through an extremely complex intertwining of the protein structures. Despite many years of study, the molecular mechanisms and determinants of transcriptional and replicative activities of the flu RdRP are still not fully understood. Nevertheless, the recent publication of several crystal structures of fragments of the RdRP subunits furnished important insights.26,29 Most importantly, the very recent elucidation of the atomic structures of complete fluA30 and B31 polymerase complexes bound to their vRNA promoter sequences and apo-RdRP from fluC32 led to an important breakthrough in understanding of the flu replication machinery, providing an atomic-level mechanistic insight into multiple functions of flu polymerase. A discussion on the structure and mechanisms of action of flu RNA synthesis machinery is out of the purpose of this perspective; for comprehensive reviews on the topic, see specific and recent reviews.25-28,33,34 In addition to being essential for the biological processes of virus transcription and replication, fluA polymerase also plays a crucial role in virus evolution.35,36 In particular, during the process of antigenic drift, one of the two sources of virus variation, the viral error-prone RdRP gives rise to mutations in the genes responsible for encoding the antigenic proteins HA and NA, thus generating new epidemic strains. The other source of variation is the antigenic shift that occurs during replication of two different fluA subtypes in the same cell, which mix and match segments of their genome giving rise to off-spring with new HA or NA antigens. This event may be responsible for pandemics if the majority of the population has not been exposed to the novel strain.37 Through the 6 ACS Paragon Plus Environment

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genetic processes of antigenic drift and antigenic shift, the virus gains the ability to constantly sidestep host immunity acquired from previous infections or vaccinations and to develop resistance against available antiviral agents. These issues, along with the limited therapeutic options against flu infections, makes it imperative to search for additional weapons in the form of next-generation anti-flu agents that could counteract viral drug resistance. Promising targets for novel antiviral drugs include all the components of the vRNP complex, i.e., fluA RdRP, NP, and vRNA.38-40 Among these, several unique enzymatic properties make RdRP an attractive target for the development of new antivirals.41,42 In particular, RdRP structure is highly conserved among fluA, B, and C strains,31,43 and its activity is highly host- and cell type-specific. Inhibition of PB2 capbinding, PA endonuclease, or PB1 polymerase domains are all valid means for antiviral therapy and their inhibition through small molecules has been actively pursued.44,45 An alternative and appealing strategy to inhibit these functions of RdRP is to interfere with its proper assembly using proteinprotein interaction (PPI) inhibitors. PPIs are critical regulatory events in many biological processes under both physiological and pathological conditions and, thus, represent a treasure trove of possible new therapeutics. There are several potential advantages to targeting PPIs: (i) the great variability and specificity of PPIs with respect to the active site of enzymes, which are often characterized by a high structural similarity between the human and pathogen isoforms, suggests that PPI inhibitors could be highly selective; (ii) the requirement for the simultaneous mutation of at least one residue on both proteins involved in the interaction to develop resistance while maintaining the interaction domain suggests that PPI inhibitors could be less prone to drug resistance than to inhibitors of enzyme active sites, for which resistance is conferred by a single amino acid mutation. In addition, a synergistic effect between an active site inhibitor and a PPI inhibitor of the same enzyme could achieve therapeutic inhibition at dose levels that are better tolerated by patients and reduce the probability of generating escape mutants.



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Until recently, PPIs have been considered “undruggable.” Unlike conventional targets for which endogenous substrates may be used as templates for the design of antagonists, it is extremely difficult to identify high affinity inhibitors of PPIs due to a number of particular challenges, many of which originate from the topology of the targeted binding sites:46 (i) the contact surface area is typically large, suggesting that tight binding is unlikely to be achieved by a small molecule owing to insufficient interactions, (ii) the interaction surfaces are often flat and relatively featureless, limiting contact to only one side of a small molecule, and (iii) the binding regions of PPIs are often non-contiguous, such that simple synthetic peptides or peptidomimetics may not access all of these domains. The development of PPI inhibitors is further complicated by additional issues: (i) there may be binding conformations suitable for small molecules that are invisible in a single crystal structure, as suggested by the adaptation of protein surfaces involved in PPI, (ii) traditional libraries of compounds used in high-throughput screenings (HTS) often furnish no viable hits, because PPI inhibitors and traditional drug-like compounds occupy quite different chemical spaces, and (iii) the activity of PPI inhibitors cannot be evaluated by commercially available assays, requiring the setup of appropriate assays. Finally, PPI inhibitors have been larger and more hydrophobic than typical drug-like compounds,47 limiting their potential to be made into drugs. Despite these challenges, to date, numerous PPI inhibitors have been developed for more than 40 PPIs, and inhibitors for six targets are now in clinical trials.48-50 The increasing number of biologically relevant crystallized protein–protein complexes, along with the availability of diverse experimental methods (HTS, biochemical and cellular assays, fragment-based approaches, and computer-aided approaches) that are able to discover PPI inhibitors51-53 has boosted this formerly minor area of research into a tremendously active field. Moreover, the inherency of PPI inhibitors to undruglikeness seems to be overcome by second-generation PPI inhibitors that are smaller and more efficient than their precursors, and even PPI inhibitors with no drug-like properties have been made orally bioavailable clinical candidates.54-56


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Flu RdRP, thanks to its heterotrimeric structure, is a particularly suitable target for the PPI inhibition approach. Among the three polymerase subunit interactions, PB1-PB2, PA-PB2, and PAPB1, only the latter has been reported to be inhibited by both peptides and small molecules and only two peptides have been identified as PA-PB2 interaction inhibitors.57,58 The aim of this Perspective is to describe the current status of the PA-PB1 complex inhibition research field. An overview of all the small molecule PA-PB1 heterodimerization inhibitors discovered so far will be provided for the first time; approaches used to identify the hit compounds, descriptions of the hit-to-lead evolution studies and the emerged structure-activity relationship (SAR) insights will also be described. Most PA-PB1 heterodimerization inhibitors have been identified by structure-based virtual screening (SBVS). Thus, a comprehensive analysis of the available PA-PB1 crystallographic structures will be given before a description of the inhibitors. In addition, we will focus on their hypothesized binding modes within the PA cavity based on common computational analyses and on their alignment with a pharmacophore model we have recently proposed.

2. CRYSTAL STRUCTURES OF POLYMERASE PA-PB1 SUBUNIT INTERACTION The discovery of small molecules that inhibit PA-PB1 complex formation was facilitated and prompted by two papers published in 2008, of x-ray crystal structures of the PA-PB1 complex (pdb codes: 3CM8 by He et al.59 and 2ZNL by Obayashi et al.60). By aligning the two crystal structures, we found significant similarities (Figure 3A), suggesting that they could be considered likewise suitable when used to search for PA-PB1 complex formation inhibitors. A comparison between the two x-ray structures was also performed by Liu and Yao,61 which demonstrated that they only differed in some loops of the PA section. However, these loops were far from the PA-PB1 binding region and there was no proof that they would have an impact on the function of the PA and PAPB1 binding surface.



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Figure 3. (A) Alignment of the PA-PB1 complex x-ray structures from previous publications.59,60 The x-ray structure by Obayashi et al. (pdb code: 2ZNL, dark-pink) was aligned with that of He et al. (pdb code: 3CM8, cyano) using Pymol. (B) Details of the PA fragment alignments; the most important residues for PB1 binding are visualized in stick mode. (C) Details of the alignment of the PB1 fragments (pdb code: 3CM8, green; pdb code: 2ZNL, orange); the most important residues for PA binding are visualized in stick mode.

He et al.59 published a clear description of the 2.9 Å structure of the PAC (residues 257–716) in a complex with the 25 PB1N terminal residues for the avian fluA H5N1 strain. Concerning the PB1 subunit, only the 15 PB1N terminal residues were used in the refined model. Previous reports have shown that the PB1N specifically binds to the PAC,62-64 allowing complex formation and nuclear transport.63,65,66 An analysis of the PAC subunit demonstrated that it consisted of 13 α-helices, 1 short 310 helix (η1) and 9 β strands, with several loops and turns. PB1N was reported to mainly interact with helices α8, α10, α11, and α13 of PAC (Figure 4). These four helices form a hydrophobic core and, indeed, the PAC-PB1N interactions were largely hydrophobic, although Hbonds and van der Waals forces were also detected.


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Figure 4. Structure of the PA-PB1 complex as published by He et al. In the inset, the binding of the PB1 resolved segment is highlighted.

At the residue level, a LLFL motif in PB1N (residues 7–10) interacted with the PAC hydrophobic core defined by F411 (α4), M595 (α8), L666 (α11), W706 and F710 (α13), and V636 and L640. In addition, W706 also interacted with residues V3 and N4 of PB1, while Q408 and N412 (α4) interacted with V3 and D2. Finally, Q670 (α11) was reported to interact with PB1N residues F9, V12, P13, and A14. PA residues P620 and I621 on β8 were also located in the PB1N interaction surface. Mutagenesis studies by He et al.59 demonstrated that the W706A/Q670A, L666G/F710E, L666G/F710G, and W706A/F710Q double mutations disrupted PAC-PB1N binding. Obayashi et al.60 performed a co-expression of PAC (residues 239–716) with PB1N terminal 81 residues from the fluA H1N1 strain. The structure of the complex, obtained with a resolution of 2.3 Å, revealed that the PAC consists of 13 α-helices and 9 β-strands. After refinement, 423 residues of PA were identified, and 15 residues of PB1 (from M1 to Q15) were visible in the electron density map, as for the 3CM8 structure.59 According to their analysis, M1 seemed unlikely to form strong 11 ACS Paragon Plus Environment


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interactions with PA, in agreement with what was observed by He et al.59 Residues D2 to N4 formed anti-parallel β-sheet-like interactions with I621 to E623 of PA. Concerning the H-bond interactions, the backbone carbonyl oxygen atoms of D2, V3, F9, L10, and V12 in PB1 interacted with E623, Q408, W706, Q670, and R673 in PA, while the backbone nitrogen atoms of D2, V3, N4, L8, and A14 in PB1 interacted with E623, N412, I621, P620, and Q670, respectively. Regarding the hydrophobic interactions, P5 packed between F411 and W706, and L8 interacted with the side chains of M595, W619, V636, and L640. In summary, by comparing the two crystal structures described by He et al. and by Obayashi et al., a number of differences were noted on the key interactions cited by the authors despite significant similarities in the structures. In particular, PA residues L666 and F710 were only mentioned by He et al.,59 while residues E623, I621, P620, W619, R673, T618, and E617 were only mentioned by Obayashi et al.60 According to the two analyses, PB1 P5, L7, K11, and P13 were involved differently. On the other hand, the contribution of the PA residues W706, Q408, N412, L640, M595, V636, and Q670 resulted of primarily importance in both studies. To analyze the two crystal structures using an identical approach, Figure 5 shows the main PAC-PB1N networks identified by the LigPlot+ program67 (version 1.4.5) for the two crystal structures 3CM859 and 2ZNL60. From a general inspection, the high similarity between the two structures is confirmed. Indeed, the specific interaction given by the PAC residues N412, Q408, E623, W706, I621, R673, and Q670 are conserved. In both the structures, also T618 results to play a role in the PAC-PB1 network, but in the structure by He et al. it is involved in hydrophobic interactions (Figure 5A), while for the Obayashi et al. structure a H-bond interaction with K11 is proposed (Figure 5B). A second possible H-bond interaction is described between K11 and E617 for the Obayashi et al. structure, suggesting that the involvement of PB1 residue K11 is not so clearly defined.


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Figure 5. LigPlot diagrams of PAC-PB1N interaction networks for crystal structures published by He et al. (A) and by Obayashi et al. (B). PAC residues are visualized in bold style (black and green colors are used to distinguish hydrophobic and H-bond interactions, respectively). PB1 residues are visualized in blue-italic style.

Two years after the publication of these x-ray structures, an interesting molecular dynamic (MD) study by Liu and Yao61 provided novel insights into PA-PB1 binding. The authors used the 3CM8 x-ray structure to run an 8 ns MD simulation of the PA-PB1 subunits, combined with molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and molecular mechanics generalized Born surface area (MM-GBSA) computations, and virtual alanine scanning. According to their studies, hydrophobic interactions provided the driving force for binding. Liu and Yao identified three hydrophobic pockets. The first one was composed of W706 and F411 from PA, which was responsible for the binding of P5 from PB1. The second hydrophobic pocket was formed by F710 and L666 and was is involved in the binding of F9 from PB1. Finally, L640, V636, M595, and W619 defined the third hydrophobic pocket, which interacts with L8 of PB1 (Figure 6). 13 ACS Paragon Plus Environment


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Figure 6. Three hydrophobic pockets in the PA cavity from 3CM8, as defined by Liu and Yao.61 First pocket, yellow; second pocket, pink; third pocket, green. PA and PB1 residues are labelled in black and red, respectively.

Based on data obtained from the pair interaction analysis and virtual alanine scanning, residues of PA reported to have a large contribution to the binding process were: W706, F710, N412, Q408, F411, L666, W619-Q623, Q670, L667, M595, T639, and L640. These results are in agreement with mutagenesis studies published by He et al.59 and Obayashi et al.60 More recently, Cusack and co-workers published the crystal structure of the complete heterotrimeric flu polymerase in two consecutive papers.30,31 In the paper by Pflug et al.,30 the crystal structure of the complete heterotrimeric polymerase of bat-specific fluA virus (bat fluA), which is evolutionarily close to human/avian fluA strains, bound to the vRNA promoter was described (pdb code: 4WSB). In the paper by Reich et al.,31 two different crystal forms of flu polymerase from the fluB/Memphis/13/03 strain were obtained (pdb codes: 4WSA and 4WRT). While reviewing this perspective, the same research group described the crystal structure of apopolymerase from the fluC/Johannesburg/1/1966 strain, revealing a new closed conformation (pdb: 5D98 and 5D9A).32 14 ACS Paragon Plus Environment

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Focusing on the PA-PB1 complex for purposes of this paper, Cusack and co-workers reported that the PA-PB1 interactions were more extensive than the crucial but limited ones described by He et al. and Obayashi et al., with a total buried surface of 17 300 Å2. In particular, the whole bat fluA polymerase crystal structure30 revealed that, aside from the interaction between the 15 PB1N and the large PAC domain (residues 258-714), the PA-linker (residues 196-257) that connects the PAN terminal endonuclease and the PAC domains wrapped around the external face of the PB1 fingers and palm domains. Although the crystallization of the complete heterotrimeric flu polymerase is a milestone in the elucidation of this target, drug discovery studies targeting the PAC cavity reported so far retain their validity. Indeed, by aligning the PA protein from structures 4WSA, 4WSB, and 4WRT published by Cusack and co-workers to the PAC subunit reported by He et al.,59 it becomes evident that the PAC cavity interacting with the PB1N terminus (1-15) displays high similarity across the structures. The structural alignment performed with Pymol68 resulted in RMSD values equal to 0.721 Å, 1.093 Å and 1.162 Å for 4WSB, 4WSA, and 4WRT, respectively (Figure 7).



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Figure 7. Aligning the PA protein from structures 4WSB (yellow), 4WSA (blue), and 4WRT (red) and the PAC from structure 3CM8 (cyano).

3. POLYMERASE PA-PB1 SUBUNITS INTERACTION INHIBITORS Anti-flu compounds that are able to interfere with the heterodimerization of PA-PB1 subunits may have major advantages over other classes of anti-flu compounds. In particular, RdRP structure is highly conserved among fluA, B, and C strains,43 suggesting that PA-PB1 heterodimerization inhibitors may be active against major subtypes of fluA and B viruses. Moreover, the residues of both PB1 and PA that are crucial for subunits interaction are highly conserved among fluA strains,61,62 and compounds that inhibit this interaction are expected to possess antiviral activity against several human and animal fluA strains. Most importantly, such inhibitors could be less prone to drug resistance, thus overcoming the main limitation that characterizes the currently available treatment.

Low propensity of PA-PB1 heterodimerization inhibitors to develop resistance The emergence of resistance against both classes of anti-flu M2 and NA inhibitors has been well documented, rendering them ineffective against most circulating fluA strains.14 In particular, 100% of fluA H3N2 and more than 95% of fluA H1N1pdm09 strains are resistant to adamantanes,15,69-71 thanks to a triple amino acids deletion at residues 28-31 or single amino acid substitution at residues 26-34 in the transmembrane domain of M2.72,73 Among the last, L26F, V27A, and S31N are the three major mutations, with the latter that is the prevailing alteration presented by more than 95% of resistant viruses.74,75 Resistance to NA inhibitors is less frequent than adamantanes,76 with a low rate of resistance to oseltamivir (1%-3%) and rare resistance to zanamivir (10,000 nM). Since positions 2 to 4, 11, and 12 of PB1 can be exchanged only by specific residues, the core PA-binding domain should be extended to aa 2 to 12. Moreover, the authors identified the exchangeable aa positions in PB11-15A outside the core, which were confined to aa 1 to 3, 6, and 13 to 15, by screening a library of 300 PB11-15A peptide variants. The variants were generated by substituting each aa residue with all 20 natural aa. Four of the five affinity enhancing mutations (V3Y, T6Y, P13K, A14I, and Q15F, 19 ACS Paragon Plus Environment


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Table 1) were located outside the core-binding region, confirming the important role of aa outside of this region. Eleven selected peptides were also evaluated in an ELISA assay, and 9 peptides showed IC50 values 1.6- to 7-fold lower than that of PB11-15A. Finally, the combination of all the five affinity-enhancing mutations resulted in a 27-fold improvement in binding affinity to PA compared to PB11-15A, as shown by a SPR binding study.

Table 1. Amino acid sequences and abilities of PB1 peptides reported by Schwemmle’s group to compete with PA binding. PA fluA, IC50 (nM)

PA fluB, IC50 (nM)

ADVNPTLLFLKVPAQNAISTTFPYT

1.80 ± 0.49

-

PB11-15A

MDVNPTLLFLKVPAQ

43.32 ± 5.31

> 3000

PB11-15B

MNINPYFLFIDVPIQ

> 3000

45 ± 12.5

PB11-15AT6Y

MDVNPYLLFLKVPAQ

21.64 ± 1.48

107.1 ± 31.3

PB15-11A

PTLLFLK

> 10,000

-

PB11-15AV3Y

MDYNPFLLFLKVPAQ

6.3 ± 2.1

-

PB11-15AP13K

MDVNPFLLFLKVKAQ

16.6 ± 5.0

-

PB11-15AA14I

MDVNPFLLFLKVPIQ

21.7 ± 9.3

-

PB11-15AQ15F

MDVNPFLLFLKVPAF

13.9 ± 2.8

-

Peptides

Amino acid sequence

PB11-25A

IC50 values represent the 50% inhibitory concentration of PB1 peptides by competitive ELISA.

3.3. Small molecules In 2008, the publication of the two PA-PB1 crystallographic structures59,60 amenable to modeling studies permitted the identification of small molecule inhibitors of this PPI.

Identification of hit compounds 1-5 by SBVS. Our group has identified many of the small molecule PA-PB1 complex formation inhibitors reported to date,88 thanks to an initial SBVS on three million compounds using the crystallographic 20 ACS Paragon Plus Environment

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structure published by He et al.59 as a template. In particular, the Fingerprints for Ligands and Proteins (FLAP) approach89 was applied to the PA cavity obtained by the 3CM8 structure and to additional conformations of PA generated by MD simulations.88 Five of the 32 preliminary compounds that emerged as candidates, which are shown as derivatives 1-5 (Figure 8), inhibited the PA-PB1 complex formation to different extents in a dose-dependent manner (IC50 = 30.4, 200, 90.7, 25.4, and 171 µM, respectively) when evaluated for their abilities to inhibit the interaction between PA and a PB11-15-Tat peptide in vitro using an ELISA-based assay. The compounds did not show significant cytotoxicity up to concentrations of 250-1000 µM in MDCK cells. When analyzed for their ability to inhibit the catalytic activity of the fluA RNA polymerase by a minireplicon assay, treatment of transfected cells with compounds 1 and 4 resulted in the dose-dependent inhibition of luciferase activity (IC50 = 18 and 31 µM, respectively) while compounds 2, 3, and 5 exhibited weak inhibitory activity at the highest concentrations (IC50 >100 µM). Compound 1 exhibited antiviral activity against a panel of fluA and B strains with EC50s ranging from 12 to 22 µM in a PRA (MDCK cells), but not against other RNA viruses or DNA viruses, demonstrating a selective profile. Accordingly to the anti-polymerase activity, compound 4 showed a low anti-flu activity (EC50 = 75 µM), while compounds 2, 3, and 5 were inactive up to 100 µM. The small molecule PAPB1 heterodimerization inhibitors identified in this study were definitely promising hit compounds thanks to their innovative mechanism of action. However, they were endowed with weak anti-PAPB1 activities, and only the most active (compound 1) could inhibit viral growth. Thus, the hit-tolead optimization was a mandatory process to increase their abilities to inhibit the PA-PB1 complex formation and, above all, acquire anti-flu activity. The hit compound 1 has been patented90 but not fully explored, while derivatives 3, 4, and 5 were objects of further structural optimization.



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Figure 8. Structures and activities of hit compounds 1-5 identified by SBVS.88

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a

The IC50 value

represents the compound concentration that reduces the PA-PB1 complex formation by 50% (ELISA assay); b the EC50 value represents the compound concentration that inhibits 50% of fluA replication (PRA assay); c the IC50 value represents the compound concentration that reduces by 50% the activity of fluA virus RNA polymerase (minireplicon assay); d the CC50 value represents the compound concentration that inhibits 50% of cell growth (MTT assay).

Cycloheptathiophene-3-carboxamide derivative 3 analogues. Hit compound 3 was a weak PA-PB1 heterodimerization inhibitor with no antiviral activity and consisted of a tetrahydrocycloheptathiophene ring bearing amide moieties functionalized with an aromatic ring at both the C-2 and C-3 positions. The study mainly focused on modifying the ofluorophenyl ring at the C-2 position91 (Figure 9). A few modifications also involved reducing the size of the cycloheptane ring and replacing the C-3 pyridine amide moiety with smaller groups. 22 ACS Paragon Plus Environment

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Many of the 35 synthesized compounds were more potent PA-PB1 complex formation inhibitors than hit compound 3; derivatives 6 and 7, characterized by a p-chlorophenyl ring and no substituent at the C-2 position, respectively, were the most potent and had IC50 values of 32 and 35 µM (ELISA assay) that were identical to that of the reference Tat-PB11-15 peptide (IC50 = 35 µM). These activities translated well to the inhibition of fluA polymerase activity (EC50s = 10 and 14 µM, respectively, in a minireplicon assay). All the compounds that were able to inhibit PA-PB1 complex formation were also active in inhibiting fluA growth (A/PR/8/34 strain, MDCK cells), with EC50 values ranging from 18 to 61 µM without showing cytotoxicity at concentrations up to 250 µM (MDCK and HEK293T cells). The best PA-PB1 heterodimerization inhibitors, compounds 6 and 7, maintained the same activity against a number of clinical isolates of fluA, including an oseltamivirresistant strain and fluB. Compound 7 is devoid of one of the aromatic moieties that seemed essential for the target recognition but demonstrated unexpected activity. Computational studies suggest that, in order to fulfill critical interactions within the PA cavity, the molecule assumes a flipped orientation with respect to compound 6 (for further details, see section 4.1). Although the activity of this class of compounds has been enhanced, particularly for the anti-flu activity (from EC50 > 100 to 18 µM, for compounds 3 and 6, respectively), a suitable potency has not yet been reached. To this end, while the C-2 position has been largely studied, additional chemical efforts will be required in order to acquire SAR insights for the C-3 pyridine and cycloheptathiophene moieties, which, on the contrary, have scarcely been explored.



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Figure 9. Structures and activities of hit compound 3 and the two best analogues, compounds 6 and 7.91 For the definitions of IC50s, EC50, and CC50 values, see Figure 8.

Pyrazolopyrimidine-3-carboxamide derivative 4 analogues. The optimization of hit compound 4 initially entailed the synthesis of a few analogues and aimed to investigate the effects of modifying the C-7 difluoromethyl, C-3 cyclopentathiophene amide, and C5 phenyl moieties attached to the pyrazolo[1,5-a]pyrimidine core (Figure 10).92 Modification of the scaffold itself was also investigated by synthesizing compounds characterized by a simple pyridine ring or a triazolopyrimidine nucleus.. In the same work, a GRID molecular interaction fields (MIFs)-based scaffold-hopping study with FLAP using hit 4 as template was also performed to search for new molecules characterized by similar chemical features on a different scaffold. Four selected compounds were acquired and tested, leading to elect a new hit-compound for the development of five additional polyamido analogues. Both approaches furnished interesting compounds, such as derivatives 8 and 9, which resulted in the highest inhibitory activities of the PA-PB1 heterodimerization with IC50s of 7.5 and 9.2 µM (ELISA assay), respectively, without interfering with unrelated PPIs. This activity correlated well with their abilities to interfere with fluA polymerase activity (EC50 = 9.2 and 17 µM, in a minireplicon assay), fluA replication (A/PR/8/34 strain-infected MDCK cells) (EC50 = 23 and 30 µM), and the growth of a panel of fluA 24 ACS Paragon Plus Environment

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and fluB strains including an oseltamivir-resistant strain (EC50s ranging from 20 to 43 µM, in MDCK cells) and were non-toxic in the cell lines used for testing. Preliminary absorption, distribution, metabolism, and excretion (ADME) studies performed on compounds 8 and 9, showed that compound 8 had water solubility of 2.4 µg/ml, low cell permeability, and metabolic stability in human liver microsome (HLM). On the other hand, although compound 9 was endowed with medium/high cell permeability, it had low water solubility (1.0 µg/ml), and its benzensulfonamide moiety underwent aromatic hydroxylation when evaluated in HLM after 30 min incubation. Hit compound 4 exhibited similar but more marked behavior. In summary, the pyrazolopyrimidine-3-carboxamide nucleus does not appear particularly suitable to impart potent PA-PB1 inhibition. On the other hand, the replacement of the pyrazolopyrimidine core with a triazolopyrimidine furnished compound 8, which emerged as a new particularly attractive PA-PB1 complex formation inhibitor. Being the only example of this chemotype within the work, it may be object of intensive structural optimizations thanks to the multiple sites offered by its structure. The hit-to-lead optimization phase should also be addressed to improve its cell permeability.



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Figure 10. Structures and activities of hit compound 4 and two of the best analogues, compounds 8 and 8.92 For the definitions of IC50s, EC50, and CC50 values, see Figure 9; the solubility value is the equilibrium solubility in aqueous media; the Peff value is the effective permeability; the membrane% value is the percentage of compound trapped into the membrane; the metabolic stability is the stability of the compound after 30 min exposure to cytochrome metabolism in human liver microsome: stable = no detection of metabolites; unstable = detection of metabolites.

Triazolopyrimidine-2-carboxamide derivative 5 analogues. Derivative 5 was the last of the five hit compounds to be investigated; optimization of compound 5 was challenging because it was one of the weakest compounds identified by the SBVS.93 The study entailed the synthesis of 28 analogues and initially focused on modifications of the phenyl ring at


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the C-2 position of the 4,7-dihydrotriazolopyrimidine nucleus, in which alkyl substituents with different chemical/physical properties, length, and steric hindrance, or aromatic substituents were placed (Figure 11). Following indications from the pharmacophore studies (see section 4.2) that a more planar and linear shape of the compounds could increase the PA-PB1 heterodimerization inhibitory effect, the 4,7-dihydrotriazolopyrimidine nucleus was oxidized, and the C-7 phenyl and C-5 methyl groups were exchanged. The amide linkage was also inverted in two aromatic compounds. Unfortunately, only two of the synthesized compounds had the ability to inhibit the PA-PB1 complex formation correlated with viral growth inhibition (EC50 and IC50 values in the range of 20-40 µM), while 12 compounds showed only one of the two activities. In the same study, two hybrid molecules, compounds 10 and 11, were also synthesized by merging the 4,7triazolopyrimidine nucleus with the cycloheptathiophene scaffold, which was characteristic of hit compound 3. Both compounds exhibited promising activity. Compound 10 had an IC50 = 1.1 µM and is the most potent of the PA-PB1 small molecule inhibitors developed thus far. In order to confirm the mechanism of action suggested by the ELISA assay, the ability of compound 10 to affect the polymerase PA-PB1-PB2 complex formation was also evaluated in a cell-based assay. In particular, the subcellular localization of the PB1 and PA subunits were determined, based on the knowledge that the PA-PB1 heterodimerization is also important for the nuclear import of the subunits and in turn their interaction with PB2, which enters the cell nucleus independently. Treatment of PA-PB1-PB2-coexpressing HEK 293T cells with compound 10 resulted in a reduced nuclear localization of PA, suggesting its ability to affect PA-PB1 binding in the cell cytoplasm and consequently blocking the intranuclear translocation of PA. This ability also translated to good antipolymerase activity (EC50 = 12 µM, in a minireplicon assay) and in broad anti-fluA and -fluB activity (EC50s ranging from 7 to 25 µM, in MDCK cells) without showing any cytotoxicity up to concentrations of 250 µM. Compound 11 exhibited similarly broad antiviral activity and had a similar cytotoxic profile with EC50 values ranging from 5 to 14 µM against fluA and B strains, even when endowed with a lower ability to inhibit the PA-PB1 complex formation (IC50 = 28 µM). 27 ACS Paragon Plus Environment


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Computational studies provided insights into the different anti-PA-PB1 activities of the two positional isomers 10 and 11, suggesting that the molecules shifted their orientations within the PA cavity and had different extents of hydrophobic and H-bond interactions (for further details, see section 4.1). Preliminary studies of their ADME profiles showed that both compounds suffered from low water solubility (< 1.0 µg/ml). In depth studies revealed that, while hit compound 5 as well as other triazolopyrimidine-based compounds were endowed with a good pharmacokinetic and solubility profile, cycloheptathiophene-based derivatives were all characterized by a very low solubility, suggesting that the latter moiety could have been responsible for the low solubility of the hybrid compounds. Hit compound 5 was not improved through the synthesis of numerous strict analogues. However, the hybrid molecule approach successfully furnished the most promising compounds 10 and 11. As shown above for compound 8, which is structurally very similar to hybrid molecules 10 and 11, this new chemotype opens the way to numerous modifications, which will be necessary to improve both solubility and cell permeability. The replacement of the cycloheptathiophene moiety with different rings characterized by a lower hydrophobicity but still able to interact with W706 within the first hydrophobic pocket, could address the above issue.


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Figure 11. Structures and activities of hit compound 5 and two of the best analogues, the hybrid molecules 10 and 11.93 For the definitions of IC50s, EC50, and CC50 values, see Figure 8; for the definitions of solubility, Peff, membrane% values, and metabolic stability, see Figure 10.

Identification by HTS and evolution of benzofurazan derivative 12. In 2013, another Italian research group published the identification of several small molecule PAPB1 complex formation inhibitors using an HTS approach (Figure 12).94 In particular, 15,000 molecules were subjected to a prescreen binding assay to the PA subunit (ELISA assay). A successive PRA on MDCK infected (A/WSN/33 strain) cells led to the identification of benzofurazan hit compound 12, which inhibited viral replication at a micromolar level (EC50 = 5 µM) despite showing cytotoxicity (CC50 = 20 µM). In order to explore the chemical space around the benzofurazan core, delineate the SAR, and increase the activity of hit compound 12, a series of 26 analogues bearing different substituents at C-4 and C-7 positions of the benzofurazan core were synthesized. One of the compounds, derivative 13, had a higher anti-fluA activity (EC50 = 1-2.5 µM) than hit compound 12, and approximately 10 compounds showed comparable or slightly lower antiviral activity levels (EC50s ranging from 5 to 40 µM). However, all the compounds maintained a certain degree of cytotoxicity (CC50s ranging from 20 to 80 µM). The ability of the compounds to disrupt the polymerase PA-PB1 subunit interaction was not reported. In a subsequent study, the same authors published a large series of benzofurazan derivatives belonging to five families, designed to explore the chemical space around the heterocyclic nucleus (Figure 12).95 In particular, the effects of modifying the C-4 and C-7 positions of the benzofurazan core was investigated further, though modifications also focused on improving the solubility of the compounds. In order to investigate the importance of the core, the benzofurazan was also replaced by isosteric heteroaromatic rings. Numerous compounds exhibited interesting anti-PA-PB1 inhibition or good anti-flu (A/WSN/33 strain MDCK infected cells) activity; however, few compounds showed a correlation between the two activities. The best anti-PA-PB1 activity was 29 ACS Paragon Plus Environment


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demonstrated by derivative 14 (IC50 = 15 µM), characterized by an EC50 = 10 µM in inhibiting viral growth. The highest anti-flu activity (EC50 = 1 µM) was shown by two compounds, with derivative 15 displaying anti-PA-PB1 activity with an IC50 = 35 µM. Unfortunately, these interesting compounds also showed a cytotoxic effect (CC50s = 20 and 40 µM, respectively). Thus, although some of the components of this class of compounds showed good anti-flu activity, the numerous structural modifications carried out in different parts of the molecule did not decrease the cytotoxicity, highlighting the unsuitability of the benzofurazan core to achieve safe PA-PB1 heterodimerization inhibitors.

Figure 12. Structures and activities of hit compound 12 identified by HTS and three of the best analogues, compounds 13-15.94,95 For the definitions of IC50, EC50, and CC50 values, see Figure 8.

Identification of 3-cyano-4,6-diphenyl-pyridine compounds 16 and 17 by SBVS. In the same year, the same research group published another work, which entailed a SBVS study aimed to identify new chemotypes that could affect PA-PB1 heterodimerization (Figure 13).96 In particular, a protein assembly built from pdb IDs C3M8 and 2ZNL95 was used to perform an MD simulation to identify the hot spot residues at PPI. Then, a commercial database of 703,200 compounds was virtually screened using a high throughput docking approach (a consensus docking 30 ACS Paragon Plus Environment

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protocol that combines Glide97 and Gold98) to search for small molecules that could mime the interactions made by the first (residues 1, 3-5) or central portion (residues 7-10) of PB1. A selection of 115 compounds was evaluated in an ELISA assay, and five compounds characterized by a 3cyano-4,6-diphenyl-pyridine nucleus were identified as weak inhibitors of the PA-PB1 complex formation (IC50s from 80 to 180 µM). Starting from these structures, a search for analogs using the web-site eMolecules yielded ten derivatives. The two compounds 16 and 17 inhibited the PA-PB1 heterodimerization with an IC50 of 35 and 30 µM, respectively, at non-toxic concentrations (CC50s = 100 - 150 µM in MDCK cells). Unfortunately, the potential for these compounds to be used as PA-PB1 complex formation inhibitors is unclear due to their very limited biological characterization, including the anti-flu activity and cytotoxicity. In order to explore the SAR of 3-cyano-4,6-diphenyl-pyridine compounds, in a successive study, 15 analogs were synthesized by either removing or replacing molecule moieties (Figure 13).99 Some of them showed interesting anti-flu activity (A/PR/8/34 strain-infected MDCK cells), with compounds 18-20 that displayed the best EC50s values (EC50s = 9.2, 7.3, and 26 µM) at no-toxic concentrations (CC50s > 250, 150, > 250 µM, MDCK cells). However, a marked lower ability to inhibit PA-PB1 complex formation (IC50s = 175, 52.6, and > 200 µM, ELISA assay) was observed. Because these compounds suffered for low solubility, the ELISA assay was repeated by using a serum-free medium. Some of them recovered the ability to inhibit PA-PB1 heterodimerization with derivatives 18-20 that showed IC50s of 35, 40, and 64 µM, respectively. Nevertheless, a strict correlation between anti-PA-PB1, anti-flu, and anti-fluA polymerase (EC50s = 86, 30, and 22 µM, in a minireplicon assay) activities did not exist. This behavior suggested that other mechanisms of action unrelated to PA-PB1 heterodimerization may contribute to the antiviral activity of the compounds. Docking and MD simulations within the PA cavity suggested for compounds 18-20 the same binding mode as reported for compounds 16 and 17;96 only the moiety at the C-3 position of the nucleus was hypothesized to bind with the PA cavity thorough different interactions.



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In summary, although the optimization process has not provided PA-PB1 inhibitors more potent than the hit compounds, some of the 3-cyano-4,6-diphenyl-pyridines exhibited interesting antiviral activity and a good cytotoxicity profile. Although experimental ADME evaluation was not performed, also this class of compounds seems to suffer from low water solubility.

Figure 13. Structures and activities of compounds 16 and 17 identified by SBVS96 and three of the best analogues, compounds 18-20.99 For the definitions of IC50s, EC50, and CC50 values, see Figure 8.

Two additional papers have reported small molecules as PA-PB1 heterodimerization inhibitors. Identification of derivatives 21-23 by SBVS. Using the crystallographic structure of PA-PB1 published by Obayashi et al.,60 Fukuoka et al. performed a SBVS using a drug database of ∼4,000 compounds (Figure 14).100 Based on the docking energy and the binding modes, 40 compounds were selected and evaluated in a PRA (A/WSN/33 strain-infected MDCK cells), leading to the identification of compounds 21 (benzbromarone), 22 (diclazuril), and 23 (trenbolone acetate), which demonstrated anti-fluA activity with EC50 values of 39, 31, and 51 µM, respectively. When the three compounds were evaluated for their binding affinities to PA, SPR results suggested a specific interaction for 32 ACS Paragon Plus Environment

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compound 21 with an estimated dissociation constant (KD) value of 48 µM, a non-specific interaction for compound 22 with a KD of 211 µM, and weak binding for compound 23 although its KD value (91 µM) was better than that of compound 22. These results were consistent with the ability of compounds 21 and 22 to inhibit fluA polymerase transcriptional activity measured using a fluA minireplicon assay, while compound 23 was ineffective in this assay system. None of these drugs has been further investigated.

Figure 14. Structures and activities of compounds 21-23 identified by SBVS.100 For the definition of EC50 value, see Figure 8; the KD value represents the dissociation constant of compound with the PA cavity.

The serendipitous identification of derivative 24. Compound 24 (AL18) is a small molecule that was serendipitously identified as a PA-PB1 complex formation inhibitor (Figure 15).101 During the evaluation of the anti-PA-PB1 heterodimerization activity of the five hit compounds identified through SBVS,88 some small molecules previously reported as inhibitors of HCMV DNA polymerase UL54/UL44 subunits interaction were included as negative controls. Unexpectedly, compound 24 also inhibited the PA-PB1 complex formation with an IC50 of 20 µM (ELISA). This inhibition translated to the ability of the compound to inhibit fluA polymerase activity with an EC50 of 16 µM in a minireplicon assay, and fluA (A/PR/8/34) and fluB (B/Lee/40) replication with EC50s of 14 and 8.3 µM, respectively (MDCK cells). Compound 24 also showed antiviral activity against several other fluA strains with EC50s ranging from 13 to 27 33 ACS Paragon Plus Environment


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µM. The compound did not exhibit cytotoxicity in MDCK cells up to at least 250 µM. To evaluate the selectivity profile of compound 24, its activity against a panel of DNA and RNA viruses other than flu and HCMV was evaluated. It was inactive against all the viruses, with some inhibitory activity exhibited against two herpesviruses (HHV-6A and HHV-6B), which may have been due to the cytotoxicity shown by the compound in the cell lines used for the assay (HSB-2 and MOLT-3, respectively). Computational studies suggested that compound 24, which is endowed with only one hydrophobic moiety, was able to compensate for lower hydrophobic interactions by establishing Hbonds (for further details, see section 4.1).

Figure 15. Structure and activities of compound 24 identified serendipitously.101 For the definitions of IC50s, EC50, and CC50 values, see Figure 8.

4.

PA-PB1

HETERODIMERIZATION

INHIBITORS:

THE

COMPUTATIONAL

APPROACHES As previously highlighted, most of the small molecules reported as PA-PB1 heterodimerization inhibitors have been identified by an initial SBVS. Computational studies supported all the hitoptimization phases, paralleling the classical medicinal chemistry approach in drug design, but also suggested new hit analogues through a scaffold hopping approach or by retrospectively analyzing the binding modes of the compounds and supporting SAR studies.


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To better determine the structural features that are essential for efficient PA-PB1 inhibition, a computational analysis of all the best inhibitors (compounds 1, 6-11, 15, 17, 21, and 24) was performed using a common approach. In particular, the predicted binding modes of the selected molecules and their alignment with a pharmacophore model were been generated using FLAP. The FLAP approach, although it is not a standard energy-based docking method, was successfully used for both virtual screening campaigns and for optimization.88,91-93 FLAP binding poses of compounds in the PA cavity from 3CM859 were also used to generate hypotheses about the key PA residues involved in the PA inhibitor interactions.

4.1. Predicted binding mode of the best PA-PB1 complex formation inhibitors within the PA cavity The FLAP poses for compounds 1,88 6,91 7,91 10,93 11,93 and 24,101 (Figure 16B-G) clearly suggested that the PA residue W706 was the primary hydrophobic interaction for the studied hits. Additional hydrophobic interactions occurred with I621 (compound 1), L666 (compounds 7 and 11), and F710 (compounds 6, 7, and 11), where compound 11 was also oriented towards the hydrophobic cavity defined by L640, V636, and W619. The pose for compound 6 was studied by using a PA conformation generated by MD with the F710 and W706 residues rotated to enlarge the second pocket, which allowed compound 6 to retain a strong π−π interaction with W706 but also to accommodate the p-chlorophenyl group in the second pocket. In addition to these hydrophobic interactions, a number of H-bond interactions have also been described to possibly stabilize the binding. One H-bond was shown for compounds 1 and 7 (with K643), for compound 10 (with Q408), and for compound 6 (with K643 or R663). Finally, three H-bonds have been proposed to stabilize compound 24 (with N412, Q408, and K643). The FLAP binding poses for compounds 8 and 9 were determined and are shown in Figure 16H and I. Compound 8 is structurally very similar to compound 11 and the two compounds also share a similar binding mode, with the triazolopyrimidine ring oriented toward the W706. As for compound 35 ACS Paragon Plus Environment


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11, efficient H-bonds were not observed. According to our approach, π−π stacking between W706 and the central phenyl moiety was observed for compound 9, with additional hydrophobic interactions between the phenyl ring located in the second hydrophobic pocket defined by F710 and L666.61 Strong double H-bonds also formed between K643 and SO2 and CO groups. The binding modes predicted for compounds 15, 17, and 21 were previously reported using different approaches. Docking studies were performed for compounds 15 and 17 using a protein assembly generated from the 3CM8 and 2ZNL structures.95,96 For compound 15, the benzofurazan scaffold was shown to interact with W706 through π−π stacking. Furthermore, an H-bond between the oxygen atom of the furazan group and Q408 and an electrostatic interaction between K643 and the nitro group were proposed. For compound 17, much like for other active compounds that possess a 3-cyano-4,6-diphenyl-pyridine nucleus, hydrophobic interactions were detected in three regions of the PA cavity, defined by: a) residues V621, C415, and F411, b) residue V628, and c) F658, F707, L666, and F710. In addition, its binding mode was stabilized by three H-bonds involving Q408, V621, and K643. In the case of compound 21, the binding pose into the PA cavity from the 2ZNL structure60 has been reported by Fukuoka et al.100 Unfortunately, the key residues for binding were not mentioned, and the authors only suggested that the compound was well placed in the pocket constructed by the α10, α4, α13, α8, and α9 segments in PA. Thus, to complete studies to understand the possible binding modes of the best 11 compounds within PA, the FLAP binding poses for compounds 15, 17, and 21 were generated; these are shown in Figure 16L-N. For compound 15, we obtained a binding mode that was similar to that reported by Pagano et al.95 For all benzofurazan derivatives, interactions with W706, K643, and Q408 were retained. For compound 17, the FLAP poses conserved the orientation of the 3-cyano-4,6-diphenylpyridine nucleus96 in correlation to W706 and the possible H-bonds with Q408 and K643. Hydrophobic interactions with the region delimited by F658, F707, L666, and F710 were also observed. However, our model showed that the third phenyl ring was oriented more towards the hydrophobic pocket generated by V636, L640, and W619 rather than towards C415. Regarding 36 ACS Paragon Plus Environment

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compound 21, the FLAP approach highlights π−π stacking between W706 and the phenyl ring and shows the possibility of H-bonds with K643 and Q408 that result from aa flexibility. Additionally, the second aromatic moiety was oriented toward the second hydrophobic pocket, as described by Liu and Yao.61

Figure 16. (A) Key residues located in the PA cavity. FLAP binding poses for compounds 1 (B), 6 (C), 7 (D), 10 (E), 11 (F), 24 (G), 8 (H), 9 (I), 15 (L), 17 (M), and 21 (N).

As shown in Figure 16, it is noteworthy that all the investigated molecules, despite their size, seem to bind the PA cavity in the hydrophobic core that corresponds to W706. The interaction of W706 with the PA inhibitors has been observed with all the docking and structure-based methods reported to date. For large molecules, additional hydrophobic interactions with the cavity generated by F710, 37 ACS Paragon Plus Environment


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L666, and F707 often occurred. According to our in silico studies, structurally similar molecules can assume a flipped orientation within the PA cavity in order to fulfill these additional hydrophobic interactions, which is clearly shown by the FLAP poses of the cycloheptathiophene-3carboxy amide analogs 6 and 7 (Figure 16C and D). In particular, compound 7, which lacks a hydrophobic moiety with respect to compound 6, moved its pyridine ring to the second hydrophobic region defined by Liu and Yao, where compound 6 placed the p-chlorophenyl ring. When additional hydrophobic interactions did not occur, favorable stabilization of the PA-small molecule interaction seemed to occur with the formation of an H-bond with Q408. This behavior was particularly evident by analyzing the binding poses of positional isomers 10 and 11 (Figure 16E and F). Indeed, while compound 11, thanks to its elongated shape, was able to recognize all the three hydrophobic regions described by Liu and Yao, the FLAP pose for compound 10 was shifted toward the opposite side of the cavity and matched only the hydrophobic region generated by W706. Compound 10 established a very favorable H-bond between its C-2 amidic carbonyl group and the Q408 residue, resulting in the most efficient PA-PB1 heterodimerization inhibition. The suggested compensation for missing hydrophobic interactions with H-bonds seems to also be confirmed by the binding mode proposed for compound 24 (Figure 16G). Indeed, this small molecule consisted only of the hydrophobic scaffold and compensated for missing π−π-stacking interactions by establishing three H-bonds. 4.2. Alignment of the best PA-PB1 complex formation inhibitors with a pharmacophore model The term pharmacophore was introduced over forty years ago102 and represents one of the most enduring concepts in the field of computer-aided drug design.103 Traditionally, a pharmacophore defined the atomic features of a molecule, disposed in a specific 3D arrangement, and was responsible for bioactivity. H-bond donors, H-bond acceptors, hydrophobic and charged groups are generally used to generate a pharmacophore. The pharmacophore approach can be considered complementary to a structure-based approach, switching perspectives from the protein to the ligand 38 ACS Paragon Plus Environment

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in the study of their interactions. Among the methods available for pharmacophore generation,104 the FLAPpharm approach has been recently described.105 According to this method, a number of selected molecules are automatically aligned and common features from this alignment are subsequently extracted.105 In the last three years, a number of pharmacophore models generated using FLAPpharm have been published, proving to be useful templates for virtual screening campaigns.106-108 Encouraged by these results, we recently published the first pharmacophore model for PA-PB1 complex inhibitors.92 The pharmacophore was automatically generated by aligning the structures of compounds 1, 6, 7, 8, and 9. The proposed model (Figure 17A) was quite planar and consisted of two hydrophobic moieties separated by a “polar belt” with two H-bond acceptor points and one Hbond donor point. The model was then validated by aligning to it other PA-PB1 heterodimerization inhibitors from the benzofurazan and 3-cyano-4,6-diphenyl pyridine series,92 such as compounds 15 and 17 in Figure 17G and H. More recently, compounds 10 and 11 were also aligned to the model93 (Figure 17I and L). In this Perspective, we also report the alignment of compounds 21 and 24 (Figure 17M and N).



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Figure 17. (A) Structure of the pharmacophore for PA-PB1 complex inhibitors generated by FLAP in pseudo-field mode;92 solid spheres represent hydrophobic (green), H-bond acceptor (red) and Hbond donor (blue) points. Alignment of compounds 1 (B), 6 (C), 7 (D), 8 (E), 9 (F), 15 (G), 17 (H), 10 (I), 11 (L), 21 (M), and 24 (N) to the pharmacophore. The hydrophobic moieties involved in the interaction with W706 are circled in light blue.

Based on the configurations presented in Figure 17, it was evident that the hydrophobic regions were all important for proper alignment, but the hydrophobic moiety involved in the interaction with W706 is variably one of the two. Concerning the H-bond donor points, they did not seem critical for interactions, as observed in previous studies.92 In addition, only the H-bond acceptor point located between the two hydrophobic moieties was preserved upon the alignment of all the structures. However, we previously discussed that H-bond interactions could occur between several groups matching these H-bond acceptor points and PA, and eight out of eleven compounds in 40 ACS Paragon Plus Environment

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Figure 17 (6, 7, 8, 9, 17, 10, 11, and 21) matched the two H-bond acceptor points. Thus, although two hydrophobic moieties and a central H-bond acceptor point seemed to be the minimum structural requirement for PA-PB1 inhibition, these additional H-bond interaction points should be exploited to achieve a more efficient PA-PB1 inhibition. In conclusion, the proposed pharmacophore model maintains its validity for the design of new PA-PB1 heterodimerization inhibitor chemotypes.

4. CONCLUSIONS The continuing threat to public health, the inadequacy of the current strategy for prophylaxis and treatment of flu infections, and continuing concerns regarding fluA pandemics dictate the need to develop novel antiviral agents. These novel agents could take advantage of steps in the virus replicative cycle that have yet to be pharmacologically exploited. Among the most promising viral targets is the RdRP, which plays a key role in the biological processes of virus transcription, replication, and evolution and is a really attractive target in the development of effective antivirals. The inhibition of a viral polymerase is a commonly used approach to identify antiviral agents, with successful examples in the anti-HIV and anti-HCV research fields. However, the development of PPI inhibitors that can interfere with viral polymerase assembly is an innovative strategy that may have major advantages; the most important advantage is a probable decrease in drug resistance. Thanks to its heterotrimeric structure formed by PA, PB1, and PB2 subunits, the flu RdRP is a challenging but particularly suitable target in pursuing a strategic PPI inhibition approach. In this context, several remarkable findings have provided the impetus behind efforts related to the discovery of small molecule PA-PB1 complex formation inhibitors that have recently enriched the literature: i) the proof of principle that was reported in 2007, that vRNA synthesis could be blocked by the specific inhibition of polymerase PA-PB1 subunit interaction by small peptides; ii) the observation that the aa of both PB1 and PA that are crucial for subunits interaction are highly conserved among fluA strains, suggesting that broad anti-fluA activity is expected by PA-PB1 heterodimerization inhibitors; iii) the evidence that mutation in the PA site leads to significant 41 ACS Paragon Plus Environment


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attenuation of the mutant viruses and that the flu possesses a limited ability to compensate for mutations within the PB1N terminus; and above all, iv) the 2008 publication of two crystal structures of fluA PA-PB1 interaction, which paved the way for computational studies. Since 2012, ten studies have reported the identification of small molecule PA-PB1 complex formation inhibitors though different approaches, i.e., SBVS, HTS, hit-evolution studies, and serendipity. The best compounds were able to inhibit PA-PB1 subunits interaction in the low micromolar range, which translated to their ability to inhibit flu polymerase and viral growth in the same range. To date, with an IC50 of 1 µM, inhibitor 10 emerged as the most potent in inhibiting PA-PB1 heterodimerization, and this mechanism of action was also confirmed in a cell-based assay. Although there have been considerable advances in the potency of PA-PB1 heterodimerization inhibitors, further efforts are required to improve the potency of these compounds up to a nanomolar range as well as their ADME profile, especially with regard to solubility. Concerning the last issue, important information emerged from analyses of the predicted binding modes of the best inhibitors and their alignment with a pharmacophore model. In particular, while three hydrophobic interactions seemed initially necessary for an efficient small molecule-PA interaction, two hydrophobic interactions now seem sufficient including a required interaction with W706. Moreover, the discovered PA-PB1 heterodimerization inhibitors were able to compensate for the lack of hydrophobic interactions by establishing one (with Q408) or more favorable H-bonds. Only the crystal structures of the PA cavity in complex with the PA-PB1 heterodimerization inhibitors can confirm their hypothesized binding modes, but the computational findings clearly suggest that prospective optimization studies aimed at obtaining more hydrophilic compounds should entail the removal of unnecessary hydrophobic groups while exploiting polar groups to establish H-bonds. Once more potent and drug-like compounds will be obtained, in vivo studies will be mandatory to determine the real potential of PA-PB1 heterodimerization inhibitors as clinical drug candidates.


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The recent elucidation of the complete structure of heterotrimeric flu polymerase from flu A, B, and C30-32 represents a crucial step forward in the design and identification of novel PPI inhibitors, allowing investigations to extend in silico to possible new regions of the polymerase complex. Moreover, the advent of innovative experimental methods, such as the recently developed RNA hybridization assay by using the entire RNP complex,109 which enables the screening of compounds, the characterization of molecules with dual functionality, and the evaluation of drug combinations, provides additional means for the identification of polymerase inhibitors. Overall, the inhibitors described here are still very far from being considered lead candidates. Although the most interesting inhibitors are non-toxic, they are not potent enough and not possess proper drug-like properties. Nevertheless, the insights outlined in this Perspective may be exploited to design further potent drug-like PA-PB1 heterodimerization inhibitors, thus boosting this anti-flu research field where, in our opinion, there is still ample room for improvement.

AUTHOR INFORMATION Corresponding Authors *S.M.: phone, +39 075 585 5146; fax, +39 075 585 5115; email, [email protected]. *L.G.: phone, +39 075 585 5632; fax, +39 075 45646; e-mail, [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have approved the final version of this manuscript. Biographies Serena Massari is an Assistant Professor in the Department of Pharmaceutical Sciences at the University of Perugia (UNIPG) (Italy). She graduated with a degree in Pharmaceutical Chemistry and Technology (2003) and received her PhD in Medicinal Chemistry (2008) from the UNIPG. From 2008 to 2015, she worked as postdoctoral researcher at the UNIPG. Her research activities entailed the selection of new druggable targets for innovative antiviral therapies, design of new 43 ACS Paragon Plus Environment


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chemical entities with desired biological activities, development of synthetic procedures for the preparation of compounds, and SAR studies. Her scientific research, documented by 28 scientific publications in peer-review journals, has mainly focused on the identification of compounds possessing activity against viruses. Recently, she has actively been working on the identification of flu PA-PB1 heterodimerization inhibitors. Laura Goracci is an Assistant Professor in the Department of Chemistry, Biology and Biotechnology at the UNIPG (Italy). She received her PhD degree in Chemical Sciences from the UNIPG (Italy) and a PhD in Organic Chemistry from the University of Bordeaux 1 (France). She has approximately 15 years of experience in the study of the structure-property (or activity) relationships of organic compounds and is particularly devoted to biological and pharmaceutical applications. Since 2008, she has been working in the fields of cheminformatics, modelling, ADMET and chemometrics, first with private companies and then at the UNIPG. She has been actively working on the identification of small molecules that inhibit PA-PB1 complex formation and has published five papers on this topic in the last 4 years. Jenny Desantis obtained her master’s degree in Pharmaceutical Chemistry and Technology at the University of Perugia (Italy) in 2013. She is currently a PhD student in Chemical and Pharmaceutical Sciences (Curriculum: Medicinal Chemistry) at the University of Perugia under the supervision of Prof. Tabarrini and Prof. Cecchetti. She is the author of one paper in the influenza research field, and her main research interests concern the design and synthesis of antiviral (influenza, HIV) small molecules. Oriana Tabarrini is an Associate Professor of Medicinal Chemistry in the Department of Pharmaceutical Sciences at the UNIPG (Italy). She graduated summa cum laude with a degree in Pharmaceutical Chemistry and Technology (1986) and then worked for several years with research grants from Pharmaceutical Industries. In 1994, she became research assistant and in 2002 was promoted to Associate Professor. Her research has mainly focused on the development of small molecules as pharmacological tools and potential chemotherapeutics. Her major research interest 44 ACS Paragon Plus Environment

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includes the identification of antiviral compounds with innovative mechanism of action. She has published over 80 research articles in leading peer-reviewed journals, including some invited reviews and patents. She has also received several project grants and is an ad-hoc reviewer for several top journals.

ACKNOWLEDGEMENTS We would like to thank G. Cruciani for carefully reading the manuscript and providing constructive comments.

ABBREVIATIONS USED aa, amino acid; ADME, absorption, distribution, metabolism, and excretion; CC50, concentration that causes a decrease of cell viability by 50%; cRNA, complementary RNA; EC50, concentration that inhibits 50% of plaque formation; ELISA, enzyme-linked immunosorbent assay; FLAP, fingerprints for ligands and proteins; flu, Influenza virus; HA, hemagglutinin; HEK293T, Human Embryonic Kidney 293T; HLM, human liver microsome; HTS, high-throughput screening; IC50, concentration that reduces the PA-PB1 complex formation by 50%; KD, dissociation constant; M1, matrix protein 1; MD, molecular dynamic; MDCK, Madin-Darby canine kidney; MIFs, molecular interaction fields; NA, neuraminidase; NP, nucleoproteins; PA, polymerase acidic protein; PB1, polymerase basic protein 1; PB2, polymerase basic protein 2; PPI, protein-protein interaction; PRA, plaque reduction assays; RdRP, RNA-dependent RNA polymerase; SAR, structure-activity relationship; SBVS, structure-based virtual screening; SPR, surface plasmon resonance; vRNA, viral RNA; vRNP, viral ribonucleoprotein.

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Influenza A Virus Polymerase Inhibitors Using the Entire Ribonucleoprotein Complex. Assay

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