*Combinational antiviral therapies for HIV*

Given that viral pathogens are absolutely dependent on the host for propagation, even more so than bacterial pathogens, research in host-directed anti-virals has advanced at a faster pace than that for anti-bacterial agents. Human Immunodeficiency virus type 1 (HIV-1), a lentivirus of the retroviral family and the causative agent of AIDS, is the most-widely stud‐ ied viral pathogen to date. HIV-1 infection causes a dramatic decline in host CD4+ T cell numbers and a progressive failure of the immune response, which makes the host suscepti‐ ble to opportunistic infections and cancer. The highly glycosylated HIV-1 envelope, in com‐ bination with the extreme diversity of circulating viral strains, have presented daunting challenges for development of an effective vaccine. Furthermore, the virus establishes chron‐ ic infection that resists the highly active antiretroviral therapy (HAART). Conventional HAART for HIV-1 infection combines three main classes of anti-viral drugs:


HAART is usually patient-specific, and its formulation is determined by the viral load and drug resistance. A traditional HAART consists of two NRTIs and a NNRTI or a PI [25]. More advanced combination therapies include a fourth class of antiretroviral drugs, HIV entry in‐ hibitors. HIV-1 entry into human cells is dependent on several sequential steps that include binding of viral envelope protein gp120 to the CD4 receptor, and conformational change in gp120 that increases its affinity to the chemokine co-receptors (CCR5 or CXCR4) and expos‐ es gp41, an HIV envelope protein that executes the fusion of HIV and host cell membranes.

Currently, there are two approved inhibitors of HIV-1 entry:


The β-chemokine receptor CCR5 was found to act as a major co-receptor for the macro‐ phage-tropic HIV-1 R5 strains, predominant in the early asymptomatic stages of virus infec‐ 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‐ lective CCR5 antagonist capable of blocking R5 HIV-1 infection *in vitro* [30].

*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‐

Given that viral pathogens are absolutely dependent on the host for propagation, even more so than bacterial pathogens, research in host-directed anti-virals has advanced at a faster pace than that for anti-bacterial agents. Human Immunodeficiency virus type 1 (HIV-1), a lentivirus of the retroviral family and the causative agent of AIDS, is the most-widely stud‐ ied viral pathogen to date. HIV-1 infection causes a dramatic decline in host CD4+ T cell numbers and a progressive failure of the immune response, which makes the host suscepti‐ ble to opportunistic infections and cancer. The highly glycosylated HIV-1 envelope, in com‐ bination with the extreme diversity of circulating viral strains, have presented daunting challenges for development of an effective vaccine. Furthermore, the virus establishes chron‐ ic infection that resists the highly active antiretroviral therapy (HAART). Conventional

**2.** non-nucleoside RT inhibitors (NNRTIs), which target the non-catalytic domain of RT,

HAART is usually patient-specific, and its formulation is determined by the viral load and drug resistance. A traditional HAART consists of two NRTIs and a NNRTI or a PI [25]. More advanced combination therapies include a fourth class of antiretroviral drugs, HIV entry in‐ hibitors. HIV-1 entry into human cells is dependent on several sequential steps that include binding of viral envelope protein gp120 to the CD4 receptor, and conformational change in gp120 that increases its affinity to the chemokine co-receptors (CCR5 or CXCR4) and expos‐ es gp41, an HIV envelope protein that executes the fusion of HIV and host cell membranes.

**2.** maraviroc, a small molecule entry inhibitor that prevents interaction between gp120

The β-chemokine receptor CCR5 was found to act as a major co-receptor for the macro‐ phage-tropic HIV-1 R5 strains, predominant in the early asymptomatic stages of virus infec‐

HAART for HIV-1 infection combines three main classes of anti-viral drugs:

vent agriculturally-important infectious disease [24].

**1.** nucleoside reverse transcriptase inhibitors (NRTIs),

Currently, there are two approved inhibitors of HIV-1 entry:

**1.** enfuvirtide, a peptide fusion inhibitor that binds to gp41 and

*Combinational antiviral therapies for HIV*

and

164 Drug Discovery

and CCR5.

**3.** protease inhibitors (PIs).

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 CD4+ 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 active and latent viral pools [41].

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 of histones in the vicinity of the integrated viral genome [42]. Thus, VA was tested as a po‐ tential agent to disrupt HIV-1 latent infection. However, years of VA treatment in combina‐ tion with HAART showed no clearance of the latent HIV reservoir [43]. A more potent HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), approved for treatment of cuta‐ neous T-cell lymphoma, was subsequently tested as a potential agent that could 'flush out' HIV-1 from latently infected cells, based on its superior effect to VA in cell culture models [44, 45]. A substantial effort has also been invested in the design and synthesis of bryostatin chemical analogs, small molecules that activate protein kinase C (PKC) with single nanomo‐ lar concentration [46]. PKC activation leads to phosphorylation of nuclear factor κB (NFκB), a key transcription factor regulator of HIV-1 gene expression [47]. However, modulation of NFκB activity requires great caution, since abnormal NFκB signaling has been related to the pathophysiology of inflammatory diseases and neurodegenerative disorders [48].

*C. elegans*, a hermaphroditic nematode normally found in soil, is a versatile, more ethicallyacceptable whole animal system for high-throughput analysis of host response to pathogen infection. *C. elegans* contains a fully sequenced genome that facilitates both genetic and ge‐ nomic analysis, offering an ideal compromise between organismal complexity and experi‐ mental tractability. *C. elegans* offers other experimental advantages, including a rapid 2-3 week life span, simple growth conditions, target-selected gene inactivation, and a relatively low cost of maintenance compared to other whole animal systems. A wealth of experimental data has demonstrated that many developmental, neurological, and biochemical processes have been highly conserved between *C. elegans* and mammals. For example, cellular func‐ tions as diverse as innate immunity, the first line of defense against pathogen infection, and RNA interference to downregulate gene expression via double-stranded RNA, are found in both *C. elegans* and higher eukaryotes, suggesting the existence of a common ancestor of these diverse species. Thus, anti-infective compounds identified using a *C. elegans* infection

Small Molecule Screens to Identify Inhibitors of Infectious Disease

http://dx.doi.org/10.5772/52502

167

*C. elegans* as a model host system has been well-studied for numerous bacterial pathogens, in‐ cluding the Gram-positive *S. aureus*, *S. pneumoniae*, and *B. thuringiensis*, and the Gram-nega‐ tive *B. pseudomallei*, *P. aeruginosa*, and *S. marcescens*. In general, different types of bacteria are fed to *C. elegans* in place of their normal *E. coli* food source to provoke detectable symptoms of

A small manual screen of 6000 synthetic compounds and 1136 natural extracts were ana‐ lyzed in an immunocompromised mutant of *C. elegans* infected with *Enterococcus faecalis* to identify compounds that promoted host survival. [51]. A total of 16 compounds and 9 ex‐ tracts were identified that either modulated bacterial growth *in vitro*, impaired pathogen vir‐ ulence, or boosted host innate immunity. Furthermore, 15 out the 16 compounds did not kill

The development of automated sorting and handling of *C. elegans* rapidly enabled highthroughput screening of small chemical libraries to identify compounds that enhanced sur‐ vival of *C. elegans* in response to bacterial infection. This methodology was enabled by the Complex Object Parametric Analyzer and Sorter (COPAS) BioSort worm sorter (Union Bio‐ metrica) to dispense a defined number of living worms into multi-well plates, which were then imaged using automated microscopy to quantify worm survival. A library of 37,200 compounds and natural product extracts was screened using the same *C. elegans*-*E. faecalis* infection system described above [52]. Twenty-eight compounds and extracts were identi‐ fied that enhanced survival of infected *C. elegans*. Six structural classes of identified com‐ pounds did not affect the growth of *E. faecalis* itself, suggesting that the small molecules inhibited a specific aspect of the host-pathogen interaction. Interestingly, two structural classes are similar to compounds previously identified in a high-throughput screen to iden‐ tify inhibitors of *P. aeruginosa* biofilm development, indicating the presence of common mo‐

A *P. aeruginosa* infection model of *C. elegans* has also been developed to screen for novel antiinfective compounds. The high-throughput assay was based on *P. aeruginosa*-induced slow

illness, such as locomotion dysregulation, intestinal cell lysis, and shortened life span.

*C. elegans* or mammalian erythrocytes, indicating that the compounds are not toxic.

lecular targets across multiple bacterial species for drug discovery [53].

model may also be translatable in humans.

*Small molecule inhibitors of bacterial infection*

A HTS of a small molecule library recently identified novel HIV latency activators [49]. The screen was performed using a lymphoma CD4+ T-cell line (SupT1) harboring latent recombi‐ nant HIV-1 and two reporters that reflect early and late virus gene expression incorporated in the HIV-1 genome [50]. A luminescent assay based on secreted alkaline phosphatase (SEAP) activity, incorporated in the late virus gene transcripts, was applied to screen a chemical library of ~200,000 compounds. Validation of 27 hits with diverse chemical struc‐ tures demonstrated induction of latent virus from various cell models. Compounds with a selective index (CC50/EC50) above 25 were chosen for downstream medicinal chemistry mod‐ ifications. Moreover, the lead compounds were shown to reactivate latent HIV from primary resting CD4+ T-cells with no induction of cell proliferation. Small molecule activators of la‐ tent HIV that act in concert using different mechanisms have a better chance of purging the virus out of infected cells [49]. Such pre-clinical data strongly suggests that successful treat‐ ment of HIV infection can be achieved only through combinational therapy consisting of di‐ verse class of antiviral drugs.
