*Screening for inhibitors of intracellular infection*

Intracellular pathogens, including viruses, parasites, and some bacteria, manipulate specific host factors in order to downregulate the host immune response or modulate host actin cy‐ toskeleton rearrangements to induce phagocytic uptake of the pathogen. *L. monocytogenes,* an intracellular Gram-positive bacteria, infects the human host primarily through ingestion of contaminated foods and causes gastrointestinal infection. In 2011, *Listeria* contamination of cantaloupes led to at least 30 deaths and ~150 illnesses in 28 states. Following internaliza‐ tion of *L. monocytogenes* in host membrane-bound vacuoles, the pore-forming cytolysin, lis‐ teriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC) mediates lysis of the vacuoles to release the pathogen into the host cell cytosol. *L. monocytogenes* then polymerizes host actin to propel itself into adjacent host cells to continue the infection proc‐ ess. To identify compounds that inhibited *L. monocytogenes* intracellular infection, a screen of 480 small molecules from the Biomol ICCB Known Bioactives library was performed using automated microscopy and image analysis [15]. Murine bone marrow-derived macrophages were infected with a GFP-expressing *L. monocytogenes* strain to assess efficiency of invasion, survival, and replication in the host. Twenty-one compounds, affecting cell functions such as actin polymerization, calcium signaling, and apoptosis, were identified that markedly de‐ creased *Listeria monocytogenes* infection efficiency. In particular, the FDA-approved anti-psy‐ chotic drug pimozide, used to treat Tourette's syndrome and schizophrenia, was shown to decrease internalization of not just *L. monocytogenes*, but other bacterial species as well, in‐ cluding *Bacillus subtilis*, *Salmonella typhimurium*, and *E. coli*. Furthermore, pimozide de‐ creased vacuole escape and cell-to-cell spread of *L. monocytogenes* in the host. Thus, pimozide is an example of a small molecule that can be re-purposed to treat infectious dis‐ ease with potential for broad spectrum anti-microbial applications.

Parasites also employ a life cycle of host cell invasion, replication, and host cell lysis during onset of infection. *Taxoplasma gondii* is the protozoan intracellular human parasite of the phylum Apicomplexa and is related to *Plasmodium* and *Cryptosporidium*, the causative agents of malaria and diarrheal disease, respectively. To discover inhibitors of *T. gondii* invasion, a high-throughput


**3. Disruption of host-pathogen interactions for novel drug discovery**

identified that target common processes in multiple pathogens [14].

sults are summarized in Table 1.

160 Drug Discovery

*Screening for inhibitors of intracellular infection*

Given the innovation gap in the discovery of novel antibiotics post-1960, strategies to inhibit novel targets are greatly needed to combat infectious disease. Multiple studies have identi‐ fied small molecule inhibitors that target gene expression of pathogen TTSS components in *P. aeruginosa*, enteropathogenic *E. coli*, and *Y. pestis* [10, 11, 12]. The small molecule virstatin, 4-[N1,8-naphthalimide)]-n-butyric acid, was identified as an inhibitor of the transcriptional regulator ToxT in *Vibrio cholerae* [13]. The small molecule, 2-imino-5-arylidene thiazolidi‐ none, which blocks TTSS-dependent functions in *S. typhimurium*, was also found to inhibit virulence in *Yersinia*, *Pseudomonas*, and *Francisella* strains, indicating that compounds can be

Research efforts have recently begun to focus on disruption of host-pathogen interactions as a new approach to identify potential targets for drug discovery, rather than solely on specif‐ ic pathogen targets or processes. In particular, the screening of small molecule libraries to identify inhibitors that block pathogen infection of the host, using such phenotypes as pathogen invasion, host morphology, and pathogen replication in the host, is a powerful ap‐ proach for therapeutic development that may uncover fundamental mechanisms of patho‐ genesis and potentially lead to discovery of new classes of anti-infective agents. Here, we describe case studies of the use of small molecules in host infection screens to identify novel inhibitors against infectious disease, including bacterial, viral, parasitic, and fungal infec‐ tions. We will discuss these studies in the context of re-purposing known drugs, inhibitor specificity, and discovery of basic mechanisms of host-pathogen interactions. The screen re‐

Intracellular pathogens, including viruses, parasites, and some bacteria, manipulate specific host factors in order to downregulate the host immune response or modulate host actin cy‐ toskeleton rearrangements to induce phagocytic uptake of the pathogen. *L. monocytogenes,* an intracellular Gram-positive bacteria, infects the human host primarily through ingestion of contaminated foods and causes gastrointestinal infection. In 2011, *Listeria* contamination of cantaloupes led to at least 30 deaths and ~150 illnesses in 28 states. Following internaliza‐ tion of *L. monocytogenes* in host membrane-bound vacuoles, the pore-forming cytolysin, lis‐ teriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC) mediates lysis of the vacuoles to release the pathogen into the host cell cytosol. *L. monocytogenes* then polymerizes host actin to propel itself into adjacent host cells to continue the infection proc‐ ess. To identify compounds that inhibited *L. monocytogenes* intracellular infection, a screen of 480 small molecules from the Biomol ICCB Known Bioactives library was performed using automated microscopy and image analysis [15]. Murine bone marrow-derived macrophages were infected with a GFP-expressing *L. monocytogenes* strain to assess efficiency of invasion, survival, and replication in the host. Twenty-one compounds, affecting cell functions such as actin polymerization, calcium signaling, and apoptosis, were identified that markedly de‐ creased *Listeria monocytogenes* infection efficiency. In particular, the FDA-approved anti-psy‐ chotic drug pimozide, used to treat Tourette's syndrome and schizophrenia, was shown to

microscopy assay was developed to distinguish between extracellular and intracellular para‐ sites in a BS-C-1 epithelial cell model, using differential labeling with fluorescent dyes [16]. Out of a 12,160 structurally-diverse small molecule library, 24 non-cytotoxic inhibitors were identi‐ fied that reduced parasite invasion to <20% compared to control wells. These molecules inhib‐ ited different aspects of the infection process, including gliding motility and secretion of host cell adhesins. One of these inhibitors, tachypleginA, was found to post-translationally modify TgMLC1, a myosin light chain component of the *T. gondii* myosin motor complex, which drives host cell penetration and parasite mobility [17]. TgMLC1 exposed to the small molecule exhib‐ ited a rapid and irreversible change in electrophoretic mobility on SDS-PAGE gels. Although the exact nature of the modification remains unclear, the modification has been mapped to amino acids V46-R59 by mass spectroscopy. These studies provide key mechanistic informa‐ tion on the importance of *T. gondii* motility in pathogenesis and illustrate the potential for small molecules to form covalent interactions with target proteins.

#### *Targeting virulence toxin mechanisms of infection*

Many Gram-negative bacteria, including *Pseudomonas* and *Yersinia*, utilize the TTSS as a pri‐ mary mechanism of virulence to inject effector proteins into the host cytosol to downregu‐ late the host immune response. A host cytotoxicity assay was designed to screen for small molecule inhibitors of *Pseudomonas aeruginosa*, a leading cause of hospital-acquired infec‐ tions in cystic fibrosis patients. *P. aeruginosa* ExoU, a TTSS effector protein, is a member of the patatin family of phospholipase A2 (PLA2) that can lyse host cell membranes during in‐ fection. A high-throughput screen of 50,000 compounds from the Chembridge Microformat Library E was performed using a colorimetric live/dead assay to identify small molecules that protected Chinese hamster ovary (CHO) cells from cytotoxicity mediated by *P. aerugino‐ sa* expressing ExoU as the sole TTSS effector [18]. A primary list of 88 compounds exhibited rescue of CHO cells from ExoU-mediated cytotoxicity. The most effective compound, pseu‐ dolipasin A, inhibited ExoU function downstream of TTSS delivery into the host. In addition to inhibition of CHO cytotoxicity, pseudolipasin A also protected the amoeba *Dictyolstelium discoideum* from ExoU-mediated killing by *P. aeruginosa* and inhibited cytotoxicity in the yeast *Saccharomyces cerevisiae* expressing ExoU. Interestingly, pseudolipasin A did not affect eukaryotic PLA2, suggesting that this small molecule may specifically target bacterial PLA2. Pseudolipasin A is representative of small molecules that do not kill or inhibit the growth of pathogens, but instead attenuate their virulence.

cell. If the YopE-β-lactamase fusion is introduced into the host, the β-lactamase will cleave the lactam ring in CCF2-AM and liberate the fluorescein, leaving the coumarin to fluoresce blue at 447nm. Using this differential fluorescence assay, 100,000 compounds from a number of sources, including the ChemDiv 2, ChemDiv 3, ChemDiv 4, Maybridge 3, Maybridge 4, and Biomol ICCB libraries, were screened for low ratios of blue-to-green fluorescence. In to‐ tal, 200 compounds were deemed potential hits, and 45 were assessed further using secon‐ dary assays, including rounded host morphology in response to *Yersinia* infection. Finally, 6 compounds were found that inhibited translocation of effectors into the host without affect‐ ing expression and function of TTSS components. Several of these compounds also inhibited host cell rounding when induced by *Pseudomonas* effectors, suggesting that these com‐

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163

A screen to identify small molecule inhibitors of *B. anthracis* also employed the CCF2-AM FRET assay. *B. anthracis*, the Gram-positive causative agent of anthrax, secretes three major toxins during infection, lethal factor (LF), protective antigen (PA), and edema factor (EF). A fusion protein between LF and β-lactamase was introduced into host cells by PA-directed endocytosis to hydrolyze the CCF2-AM fluorogenic substrate [21]. Out of 70,094 compounds tested, 1170 initial hits exhibited concentration-dependent inhibition of β-lactamase activity. Thirty compounds with known biological activities and/or were high confidence hits were selected for further analysis. Three compounds, NCGC00084148-01, diphyllin, and niclosa‐ mide, exhibited protective effects from anthrax LF, a LF fusion to *Pseudomonas* exotoxin, and diphtheria toxin in RAW264.7 murine macrophages and CHO cells, and are thought to inter‐

The interaction between *B. anthracis* EF and its cellular activator, calmodulin (CaM), became the basis of a two-step tandem screen to identify small molecule inhibitors of anthrax infec‐ tion. A library of 10,000 compounds (Chembridge, Library # ET350-1) in pools of 8 was screened to identify small molecules that blocked an EF-induced flat to round morphology change in Y1 murine adrenocortical cells [22]. Twenty-four initial hits were then individual‐ ly tested using surface plasmon resonance (SPR) to identify molecules that block interactions between EF and immobilized CaM. One compound, (4-[4-(3,4-cichlorophenyl)-thiazolylami‐ no]-benzenesulfonamide) 10506-2A, efficiently inhibited EF-CaM binding in a dose-depend‐ ent manner, and was found to specifically target the CaM binding region of EF by fluorescence spectroscopy. Since this compound was found to be toxic in cultured mammali‐ an cells, a series of structurally-related compounds was synthesized, and a new inhibitory

Discovery of small molecule inhibitors has also been extended to plant pathogen systems as an approach to develop commercially-relevant chemicals to protect crops assets from dis‐ ease. The Gram-negative pathogen *Pseudomonas syringae* expresses a TTSS, enters plant tis‐ sues through the stomata or wounds, and infects a wide range of plant species. A major challenge in the application of small molecule screens to plant-pathogen interactions is the development of high-throughput methodology with a plant model system. A high-through‐ put liquid assay was developed based on *P. syringae*-induced bleaching of *Arabidopsis thali‐*

pounds may have a broad-spectrum anti-infective effect.

compound with reduced toxicity was subsequently identified.

*Small molecule discovery in plant-pathogen interactions*

fere with toxin internalization in the host.

Inhibitors of *P. aeruginosa* virulence have also been identified using a cell-based yeast pheno‐ typic assay in combination with a large-scale small molecule screen. A total of 505 *P. aerugi‐ nosa* virulence factors and essential genes were individually overexpressed in *S. cerevisae* to downselect genes that inhibited yeast growth [19]. Nine genes strongly or partially impaired yeast growth, including three TTSS effectors, ExoS, ExoT, and ExoY. ExoS has been previ‐ ously shown to ADP-ribosylate multiple downstream targets, including vimentin, the Ras family of small GTP-binding proteins, and cyclophilin A. Given that ExoS is a critical media‐ tor of *P. aeruginosa* chronic infections, a library of 56,280 compounds was screened to find inhibitors of ExoS ADP-ribosylation activity that rescued cytotoxicity in yeast. Six com‐ pounds were identified that restored yeast growth. The most promising compound, exosin, was found to modulate ExoS enzymatic activity *in vitro* and exhibited a protective effect against *P. aeruginosa* infection in mammalian CHO cells. This study demonstrates the effec‐ tive use of a simple eukaryotic host, baker's yeast, as a tool for drug screening for applica‐ tions in controlling infectious disease in humans.

Another pathogen family that employs the TTSS is *Yersinia*, which secrete Yop effectors into the host cell. There are three *Yersinia* human pathogens, *Y. pestis*, the etiological agent of pla‐ gue via intradermal fleabites or inhalation, and *Y. pseudotuberculosis* and *Y. enterocolitica*, which cause mild and self-limiting enteric disease by the oral route. HTS strategies have been developed to identify small molecules that inhibit translocation of the Yops into host cells. A recombinant *Y. pseudotuberculosis* strain was constructed to express a chimeric pro‐ tein containing the first 100 amino acids of YopE, which contains the proper translocation signals to inject into the host, fused to a fragment of β-lactamase. [20]. This bacterial strain was used to infect HEp-2 host cells treated with a non-membrane-permeating, non-fluores‐ cent dye CCF2-AM, which fluoresces green at 520nm, as a result of intramolecular FRET be‐ tween the 7-hydroxycoumarin and fluorescein molecules, conjugated by a lactam ring. Upon cellular uptake, CCF2-AM is modified by cytoplasmic esterases and is trapped in the host cell. If the YopE-β-lactamase fusion is introduced into the host, the β-lactamase will cleave the lactam ring in CCF2-AM and liberate the fluorescein, leaving the coumarin to fluoresce blue at 447nm. Using this differential fluorescence assay, 100,000 compounds from a number of sources, including the ChemDiv 2, ChemDiv 3, ChemDiv 4, Maybridge 3, Maybridge 4, and Biomol ICCB libraries, were screened for low ratios of blue-to-green fluorescence. In to‐ tal, 200 compounds were deemed potential hits, and 45 were assessed further using secon‐ dary assays, including rounded host morphology in response to *Yersinia* infection. Finally, 6 compounds were found that inhibited translocation of effectors into the host without affect‐ ing expression and function of TTSS components. Several of these compounds also inhibited host cell rounding when induced by *Pseudomonas* effectors, suggesting that these com‐ pounds may have a broad-spectrum anti-infective effect.

A screen to identify small molecule inhibitors of *B. anthracis* also employed the CCF2-AM FRET assay. *B. anthracis*, the Gram-positive causative agent of anthrax, secretes three major toxins during infection, lethal factor (LF), protective antigen (PA), and edema factor (EF). A fusion protein between LF and β-lactamase was introduced into host cells by PA-directed endocytosis to hydrolyze the CCF2-AM fluorogenic substrate [21]. Out of 70,094 compounds tested, 1170 initial hits exhibited concentration-dependent inhibition of β-lactamase activity. Thirty compounds with known biological activities and/or were high confidence hits were selected for further analysis. Three compounds, NCGC00084148-01, diphyllin, and niclosa‐ mide, exhibited protective effects from anthrax LF, a LF fusion to *Pseudomonas* exotoxin, and diphtheria toxin in RAW264.7 murine macrophages and CHO cells, and are thought to inter‐ fere with toxin internalization in the host.

The interaction between *B. anthracis* EF and its cellular activator, calmodulin (CaM), became the basis of a two-step tandem screen to identify small molecule inhibitors of anthrax infec‐ tion. A library of 10,000 compounds (Chembridge, Library # ET350-1) in pools of 8 was screened to identify small molecules that blocked an EF-induced flat to round morphology change in Y1 murine adrenocortical cells [22]. Twenty-four initial hits were then individual‐ ly tested using surface plasmon resonance (SPR) to identify molecules that block interactions between EF and immobilized CaM. One compound, (4-[4-(3,4-cichlorophenyl)-thiazolylami‐ no]-benzenesulfonamide) 10506-2A, efficiently inhibited EF-CaM binding in a dose-depend‐ ent manner, and was found to specifically target the CaM binding region of EF by fluorescence spectroscopy. Since this compound was found to be toxic in cultured mammali‐ an cells, a series of structurally-related compounds was synthesized, and a new inhibitory compound with reduced toxicity was subsequently identified.

### *Small molecule discovery in plant-pathogen interactions*

late the host immune response. A host cytotoxicity assay was designed to screen for small molecule inhibitors of *Pseudomonas aeruginosa*, a leading cause of hospital-acquired infec‐ tions in cystic fibrosis patients. *P. aeruginosa* ExoU, a TTSS effector protein, is a member of the patatin family of phospholipase A2 (PLA2) that can lyse host cell membranes during in‐ fection. A high-throughput screen of 50,000 compounds from the Chembridge Microformat Library E was performed using a colorimetric live/dead assay to identify small molecules that protected Chinese hamster ovary (CHO) cells from cytotoxicity mediated by *P. aerugino‐ sa* expressing ExoU as the sole TTSS effector [18]. A primary list of 88 compounds exhibited rescue of CHO cells from ExoU-mediated cytotoxicity. The most effective compound, pseu‐ dolipasin A, inhibited ExoU function downstream of TTSS delivery into the host. In addition to inhibition of CHO cytotoxicity, pseudolipasin A also protected the amoeba *Dictyolstelium discoideum* from ExoU-mediated killing by *P. aeruginosa* and inhibited cytotoxicity in the yeast *Saccharomyces cerevisiae* expressing ExoU. Interestingly, pseudolipasin A did not affect eukaryotic PLA2, suggesting that this small molecule may specifically target bacterial PLA2. Pseudolipasin A is representative of small molecules that do not kill or inhibit the growth of

Inhibitors of *P. aeruginosa* virulence have also been identified using a cell-based yeast pheno‐ typic assay in combination with a large-scale small molecule screen. A total of 505 *P. aerugi‐ nosa* virulence factors and essential genes were individually overexpressed in *S. cerevisae* to downselect genes that inhibited yeast growth [19]. Nine genes strongly or partially impaired yeast growth, including three TTSS effectors, ExoS, ExoT, and ExoY. ExoS has been previ‐ ously shown to ADP-ribosylate multiple downstream targets, including vimentin, the Ras family of small GTP-binding proteins, and cyclophilin A. Given that ExoS is a critical media‐ tor of *P. aeruginosa* chronic infections, a library of 56,280 compounds was screened to find inhibitors of ExoS ADP-ribosylation activity that rescued cytotoxicity in yeast. Six com‐ pounds were identified that restored yeast growth. The most promising compound, exosin, was found to modulate ExoS enzymatic activity *in vitro* and exhibited a protective effect against *P. aeruginosa* infection in mammalian CHO cells. This study demonstrates the effec‐ tive use of a simple eukaryotic host, baker's yeast, as a tool for drug screening for applica‐

Another pathogen family that employs the TTSS is *Yersinia*, which secrete Yop effectors into the host cell. There are three *Yersinia* human pathogens, *Y. pestis*, the etiological agent of pla‐ gue via intradermal fleabites or inhalation, and *Y. pseudotuberculosis* and *Y. enterocolitica*, which cause mild and self-limiting enteric disease by the oral route. HTS strategies have been developed to identify small molecules that inhibit translocation of the Yops into host cells. A recombinant *Y. pseudotuberculosis* strain was constructed to express a chimeric pro‐ tein containing the first 100 amino acids of YopE, which contains the proper translocation signals to inject into the host, fused to a fragment of β-lactamase. [20]. This bacterial strain was used to infect HEp-2 host cells treated with a non-membrane-permeating, non-fluores‐ cent dye CCF2-AM, which fluoresces green at 520nm, as a result of intramolecular FRET be‐ tween the 7-hydroxycoumarin and fluorescein molecules, conjugated by a lactam ring. Upon cellular uptake, CCF2-AM is modified by cytoplasmic esterases and is trapped in the host

pathogens, but instead attenuate their virulence.

162 Drug Discovery

tions in controlling infectious disease in humans.

Discovery of small molecule inhibitors has also been extended to plant pathogen systems as an approach to develop commercially-relevant chemicals to protect crops assets from dis‐ ease. The Gram-negative pathogen *Pseudomonas syringae* expresses a TTSS, enters plant tis‐ sues through the stomata or wounds, and infects a wide range of plant species. A major challenge in the application of small molecule screens to plant-pathogen interactions is the development of high-throughput methodology with a plant model system. A high-through‐ put liquid assay was developed based on *P. syringae*-induced bleaching of *Arabidopsis thali‐* *ana* cotyledon seedlings, which signifies a loss of chlorophyll from plant tissues and is indicative of bacterial pathogenesis [23]. A screen of ~200 small molecules active in *Arabidop‐ sis* (LATCA, Library of Active Compounds in Arabidopsis) identified several sulfanilamide compounds, including sulfamethoxazole, sulfadiazine, and sulfapyridine, that prevented co‐ tyledon bleaching upon *P. syringae* infection. The most potent compound, sulfamethoxazole, also inhibited *P. syringae* growth in mature soil-grown plants. A similar assay was used to implicate the same compound, sulfamethoxazole, and the indole alkaloid gramine as inhibi‐ tors of *Fusarium graminearum* fungal infection in *Arabidopsis* and wheat, indicating that this strategy represents a relevant surrogate system for identification of compounds that can pre‐ vent agriculturally-important infectious disease [24].

tion, whereas the T-cell-tropic strains (using the CXCR4 co-receptor) become prevalent in the symptomatic stages concomitant with the decline of CD4+ T-cells [26]. CCR5 is an attrac‐ tive target for development of HIV-1 entry inhibitors, given the discovery that HIV-1 nonprogressors, individuals homozygous for a 32-bp deletion in the coding region of CCR5 gene (CCR5Δ32) were naturally resistant to infection with R5 HIV-1 [27]. Natural and syn‐ thetic CCR5 ligands such as RANTES, AOP-RANTES, Mip-1α, Mip-1β,and Met-RANTES were found to efficiently protect against R5 HIV-1 infection [28, 29]. Thus, the first published high throughput screen (HTS) for discovery of non- peptide inhibitors of HIV-1 entry was performed in a virus-free cell-based system using [125I]-labeled RANTES. A strong inhibitor of RANTES binding to CCR5 stably expressed on the surface of CHO cells was identified from the library of Takeda Chemical Industries. Further chemical modifications of the lead compound designated TAK-779 produced a potent (IC50 1.4 nM in CHO/CCR cells) and se‐

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165

The number of CCR5 inhibitors has significantly grown since the discovery of TAK-779, but very few compounds have entered clinical trials, and only maraviroc has been approved for clinical use [31]. A radiolabeled-chemokine binding assay similar to one applied for the identification of TAK-779 was used in a HTS of a small molecule library at Pfizer for the dis‐ covery of UK-107,543, which had become a scaffold for intensive medicinal chemistry, pro‐ ducing ~1,000 analog compounds, from which maraviroc (UK-427,857) was selected for its excellent preclinical pharmacokinetics (90% inhibitory concentration of 2 nM in pool of PBMCs from various donors) [32]. Despite its proven efficacy against HIV-1 R5 infection, maraviroc is vulnerable to gp120 escape mutations [33]. Site-directed mutagenesis and mo‐ lecular modeling studies have identified a common binding pocket on CCR5 that is shared by various small-molecule CCR5 inhibitors [34, 35, 36]. Emerging details on gp120 and CCR5 points of interaction and binding thermodynamics provide valuable information that can be applied in developing tools for rational design of novel HIV-1 entry inhibitors [37, 38]. Efficient block of HIV entry into host cells is essential to curtail virus dissemination and is a key step towards eradication of HIV infection. The current HAART regiment can reduce HIV replication to very low levels (below 50 copies/ml plasma) and can lead to recovery of

 T-cell counts but not cure the infection. Patients that have been successfully treated with HAART for years have experienced a rapid virus rebound upon termination of the therapeutic regiment [39, 40]. Such clinical cases present evidence that HIV establishes a chronic infection that resists current HAART designed to target actively replicating virus. A deliberate and controllable induction of HIV-1 replication from its latent reservoirs in com‐ bination with HAART is a novel and actively pursued approach that aims to eliminate both

Researchers often seek new anti-infective agents amongst small molecules that have previ‐ ously been approved for the treatment of cancer and neurological diseases, since they have well-established pharmacokinetics and in most cases, known molecular mechanisms of ac‐ tion. One example of this is the histone deacetylase (HDAC) inhibitor, valproic acid (VA), which had previously been approved for treatment of neurological and psychiatric disor‐ ders. HIV-1 has been shown to enter dormancy using epigenetic silencing via deaceylation

lective CCR5 antagonist capable of blocking R5 HIV-1 infection *in vitro* [30].

CD4+

active and latent viral pools [41].
