Representing high throughput expression profiles via perturbation barcodes reveals compound targets.

High throughput mRNA expression profiling can be used to characterize the response of cell culture models to perturbations such as pharmacologic modulators and genetic perturbations. As profiling campaigns expand in scope, it is important to homogenize, summarize, and analyze the resulting data in a manner that captures significant biological signals in spite of various noise sources such as batch effects and stochastic variation. We used the L1000 platform for large-scale profiling of 978 representative genes across thousands of compound treatments. Here, a method is described that uses deep learning techniques to convert the expression changes of the landmark genes into a perturbation barcode that reveals important features of the underlying data, performing better than the raw data in revealing important biological insights. The barcode captures compound structure and target information, and predicts a compound's high throughput screening promiscuity, to a higher degree than the original data measurements, indicating that the approach uncovers underlying factors of the expression data that are otherwise entangled or masked by noise. Furthermore, we demonstrate that visualizations derived from the perturbation barcode can be used to more sensitively assign functions to unknown compounds through a guilt-by-association approach, which we use to predict and experimentally validate the activity of compounds on the MAPK pathway. The demonstrated application of deep metric learning to large-scale chemical genetics projects highlights the utility of this and related approaches to the extraction of insights and testable hypotheses from big, sometimes noisy data.

Benzoxazolinone aryl sulfonamides as potent, selective Nav1.7 inhibitors with in vivo efficacy in a preclinical pain model.

Studies on human genetics have suggested that inhibitors of the Nav1.7 voltage-gated sodium channel hold considerable promise as therapies for the treatment of chronic pain syndromes. Herein, we report novel, peripherally-restricted benzoxazolinone aryl sulfonamides as potent Nav1.7 inhibitors with excellent selectivity against the Nav1.5 isoform, which is expressed in the heart muscle. Elaboration of initial lead compound 3d afforded exemplar 13, which featured attractive physicochemical properties, outstanding lipophilic ligand efficiency and pharmacological selectivity against Nav1.5 exceeding 1000-fold. Key structure-activity relationships associated with oral bioavailability were leveraged to discover compound 17, which exhibited a comparable potency/selectivity profile as well as full efficacy following oral administration in a preclinical model indicative of antinociceptive behavior.

Discovery of selective, orally bioavailable, N-linked arylsulfonamide Nav1.7 inhibitors with pain efficacy in mice.

The voltage-gated sodium channel Nav1.7 is a genetically validated target for the treatment of pain with gain-of-function mutations in man eliciting a variety of painful disorders and loss-of-function mutations affording insensitivity to pain. Unfortunately, drugs thought to garner efficacy via Nav1 inhibition have undesirable side effect profiles due to their lack of selectivity over channel isoforms. Herein we report the discovery of a novel series of orally bioavailable arylsulfonamide Nav1.7 inhibitors with high levels of selectivity over Nav1.5, the Nav isoform responsible for cardiovascular side effects, through judicious use of parallel medicinal chemistry and physicochemical property optimization. This effort produced inhibitors such as compound 5 with excellent potency, selectivity, behavioral efficacy in a rodent pain model, and efficacy in a mouse itch model suggestive of target modulation.

Association of respiratory failure with inhibition of NaV1.6 in the phrenic nerve.

As part of a drug discovery effort to identify potent inhibitors of NaV1.7 for the treatment of pain, we observed that inhibitors produced unexpected cardiovascular and respiratory effects in vivo. Specifically, inhibitors administered to rodents produced changes in cardiovascular parameters and respiratory cessation. We sought to determine the mechanism of the in vivo adverse effects by studying the selectivity of the compounds on NaV1.5, NaV1.4, and NaV1.6 in in vitro and ex vivo assays. Inhibitors lacking sufficient NaV1.7 selectivity over NaV1.6 were associated with respiratory cessation after in vivo administration to rodents. Effects on respiratory rate in rats were consistent with effects in an ex vivo hemisected rat diaphragm model and in vitro NaV1.6 potency. Furthermore, direct blockade of the phrenic nerve signaling was observed at exposures known to cause respiratory cessation in rats. Collectively, these results support a significant role for NaV1.6 in phrenic nerve signaling and respiratory function.

A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells.

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

Targeted overexpression of EZH2 in the mammary gland disrupts ductal morphogenesis and causes epithelial hyperplasia.

The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to 12 years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with beta-catenin, inducing beta-catenin nuclear accumulation in mammary epithelial cells and activating Wnt/beta-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with beta-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma.

Assembly and intracellular trafficking of HLA-B*3501 and HLA-B*3503.

Residue 116 of major histocompatibility complex (MHC) class I heavy chains is an important determinant of assembly, that can influence rates of ER-Golgi trafficking, binding to the transporter associated with antigen processing (TAP), tapasin dependence of assembly, and the efficiency and specificity of peptide binding. Here, we investigated assembly and peptide-binding differences between HLA-B*3501(S116) and HLA-B*3503(F116), two alleles differing only at position 116 of the MHC class I heavy chain, that are associated respectively with normal or rapid AIDS progression. A reduced intracellular maturation rate was observed for HLA-B*3503 in HIV-infected and uninfected cells, which correlated with enhanced binding of HLA-B*3503 to TAP. No significant differences in the intrinsic efficiency of in vitro peptide binding by HLA-B*3501 and HLA-B*3503 were measurable with several common peptides or peptide libraries, and both allotypes were relatively tapasin-independent for their assembly. However, thermostability differences between the two allotypes were measurable in a CD4(+) T cell line. These findings suggest that compared to HLA-B*3501, a reduced intracellular peptide repertoire for HLA-B*3503 could contribute to its slower intracellular trafficking and stronger association with rapid AIDS progression.

HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail.

To avoid immune recognition by cytotoxic T lymphocytes (CTLs), human immunodeficiency virus (HIV)-1 Nef disrupts the transport of major histocompatibility complex class I molecules (MHC-I) to the cell surface in HIV-infected T cells. However, the mechanism by which Nef does this is unknown. We report that Nef disrupts MHC-I trafficking by rerouting newly synthesized MHC-I from the trans-Golgi network (TGN) to lysosomal compartments for degradation. The ability of Nef to target MHC-I from the TGN to lysosomes is dependent on expression of the mu1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN to endolysosomal pathways. We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I. Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail. In sum, our evidence suggests that binding of AP-1 to the Nef-MHC-I complex is an important step required for inhibition of antigen presentation by HIV.

Human immunodeficiency virus type 1 Nef domains required for disruption of major histocompatibility complex class I trafficking are also necessary for coprecipitation of Nef with HLA-A2.

Human immunodeficiency virus type 1 (HIV-1) Nef is a critical protein that is necessary for HIV pathogenesis. Its roles include the disruption of major histocompatibility complex class I (MHC-I) and CD4 trafficking to promote immune evasion and viral spread. Mutational analyses have revealed that separate domains of Nef are required to affect these two molecules. To further elucidate how Nef disrupts MHC-I trafficking in T cells, we examined the role of protein domains that are required for this function (N-terminal alpha helix, polyproline, acidic, and oligomerization domains). We found that each of these regions was required for Nef to disrupt the transport of HLA-A2 to the cell surface and for Nef to coprecipitate with HLA-A2.

HIV-1 Nef disrupts antigen presentation early in the secretory pathway.

Human immunodeficiency virus, type 1 Nef disrupts viral antigen presentation and promotes viral immune evasion from cytotoxic T lymphocytes. There is evidence that Nef acts early in the secretory pathway to redirect major histocompatibility complex class I (MHC-I) from the trans-Golgi network to the endolysosomal pathway. However, a competing model suggests that Nef acts much later by accelerating MHC-I turnover at the cell surface. Here we demonstrate that Nef targets early forms of MHC-I molecules in the endoplasmic reticulum by preferentially binding hypophosphorylated cytoplasmic tails. The Nef-MHC-I complex migrates normally into the Golgi apparatus but subsequently fails to arrive at the cell surface and become phosphorylated. Cell type-specific differences in the rate of MHC-I transport through the secretory pathway correlate with responsiveness to Nef and co-precipitation of adaptor protein 1 with the Nef.MHC-I complex. We propose that the assembly of a Nef.MHC-I.adaptor protein 1 complex early in the secretory pathway is important for Nef activity.

Species-specific effects of HIV-1 Nef-mediated MHC-I downmodulation.

In vitro studies have revealed that human immunodeficiency virus-1 (HIV-1) Nef functionally interacts with amino acid residues in the cytoplasmic tail of major histocompatibility complex class I (MHC-I) molecules, reducing their expression on the cell surface and protecting them from cytotoxic T lymphocyte (CTL) lysis. To obtain a better understanding of Nef's effects in vivo, it would be helpful to have a mouse model system. However, it is not known whether Nef will affect murine MHC-I proteins. We find that Nef downmodulates human MHC-I HLA-A2 more efficiently than murine MHC-I molecules in HeLa cells and that Nef does not function efficiently in murine endothelial cells. Studies with chimeric molecules indicate that the MHC-I cytoplasmic tail is primarily responsible for species-specific differences. However, there are also effects attributable to the extracellular domain.

Rev activity determines sensitivity of HIV-1-infected primary T cells to CTL killing.

The HIV Nef protein is thought to promote HIV immune evasion by downmodulating MHC-I and protecting infected cells from CTL killing. In addition, we demonstrated that Rev, an HIV regulatory protein needed for expression of the HIV late genes, can influence CTL killing. When Rev activity level was reduced by virtue of amino acid alterations in the Rev protein sequence, infected cells were more resistant to anti-Gag and anti-Env CTL killing. A screen of primary viral isolates revealed that viruses derived from asymptomatic, infected people had lower Rev activity, lower Gag levels, and greater resistance to anti-Gag CTL killing. Thus, rev alleles with low activity may have a selective advantage in infected people with effective immune responses.