Summary Malaria sporozoites are rapidly targeted to the liver where they pass through Kupffer cells and infect hepatocytes, their initial site of replication in the mammalian host. We show that sporozoites, as well as their major surface proteins, the CS protein and TRAP, recognize distinct cell type‐specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not from sinusoidal endothelia. Recombinant Plasmodium falciparum CS protein and TRAP bind to heparan sulphate on hepatocytes and both heparan and chondroitin sulphate proteoglycans on stellate cells. On Kupffer cells, CS protein predominantly recognizes chondroitin sulphate, whereas TRAP binding is glycosaminoglycan independent. Plasmodium berghei sporozoites attach to heparan sulphate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulphate and heparan sulphate proteoglycans. CS protein also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver. In situ binding studies using frozen liver sections indicate that the majority of the CS protein binding sites are associated with these matrix proteoglycans. Our data sug‐gest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.

NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1-associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5, suggesting that the complex might methylate histone H3-Lys-4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys-4. The identified components form at least two distinctly sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of an NIF-1 complex of ϳ1.5 MDa and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-␣ was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes, although the separate NIF-1 and NRC complexes appear to functionally interact in the cell.A number of crucial insights into the multilayered regulation of transcription have been uncovered through study of nuclear hormone receptor-mediated gene expression. Nuclear hormone receptors are hormone-and ligand-dependent transcription factors that control the coordinated expression of gene networks in numerous physiological, developmental, and metabolic processes (1). Dysfunction of nuclear receptor signaling leads to a number of proliferative, reproductive, and metabolic diseases such as cancer, infertility, obesity, and diabetes (2). The biological functions of nuclear hormone receptors rely on coactivators that represent a diverse group of proteins that enhance nuclear receptor-mediated transcription (3). Extensive studies on the in vivo functions of nuclear receptors have led to the identification and characterization of ϳ200 coactivators, all of which have been catalogued on-line at the Nuclear Receptor Signaling Atlas. Coactivators, in general, are known to act by: (i) bridging factors to recruit additional cofactors to DNA-bound nuclear receptors, e.g. p160/SRC proteins (4, 5); (ii) exhibiting various enzymatic activities such as methylation, acetylation, and others to modulate chromatin (6 -8); and (iii) interfacing between DNA-bound nuclear receptors and the basal transcriptional machinery, e.g. TRAP/DRIP complex (9).Nuclear receptor coregulator (NRC) 3 also referred to as ASC-2, TRBP...

CCR4-NOT is an evolutionarily conserved, multicomponent complex known to be involved in transcription as well as mRNA degradation. Various subunits (e.g. CNOT1 and CNOT7/CAF1) have been reported to be involved in influencing nuclear hormone receptor activities. Here, we show that CCR4/CNOT6 and RCD1/CNOT9, members of the CCR4-NOT complex, potentiate nuclear receptor activity. RCD1 interacts in vivo and in vitro with NIF-1 (NRC-interacting factor), a previously characterized nuclear receptor cotransducer that activates nuclear receptors via its interaction with NRC. As with NIF-1, RCD1 and CCR4 do not directly associate with nuclear receptors; however, they enhance ligand-dependent transcriptional activation by nuclear hormone receptors. CCR4 mediates its effect through the ligand binding domain of nuclear receptors and small interference RNA-mediated silencing of endogenous CCR4 results in a marked decrease in nuclear receptor activation. Furthermore, knockdown of CCR4 results in an attenuated stimulation of RAR␣ target genes (e.g. Sox9 and HoxA1) as shown by quantitative PCR assays. The silencing of endogenous NIF-1 also resulted in a comparable decrease in the RAR-mediated induction of both Sox9 and HoxA1. Furthermore, CCR4 associates in vivo with NIF-1. In addition, the CCR4-enhanced transcriptional activation by nuclear receptors is dependent on NIF-1. The small interference RNA-mediated knockdown of NIF-1 blocks the ligand-dependent potentiating effect of CCR4. Our results suggest that CCR4 plays a role in the regulation of certain endogenous RAR␣ target genes and that RCD1 and CCR4 might mediate their function through their interaction with NIF-1.The nuclear receptor superfamily consists of ligand-regulated transcription factors that orchestrate the coordinated expression of gene networks in diverse physiological, developmental, and metabolic processes (1, 2).

Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2) maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2. These inhibitors should prove useful for further chromatin biology investigations.

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The PD-1/PD-L1 molecular pathway is one of the primary mechanisms of immune evasion deployed by cancer cells. Induction of PD-L1 expression on cancer cells is associated with inhibition of immune responses against cancer, thus permitting cancer progression and metastasis. Activation of PD-1/PD-L1 pathway induces apoptosis of activated T-cells, inhibits their proliferation, facilitates T-cell anergy and exhaustion and enhances the function of regulator T-cells. Therefore, blocking this pathway restores the proliferation and cytotoxicity of CTLs, inhibits the function of Tregs and results in decreased T-cell apoptosis. A number of cancer immunotherapy agents targeting PD-1/PD-L1 have been developed and approved for a number of malignancies (PD-1: Nivolumab, Pembrolizumab, PD-L1: Atezolizumab, Avelumab, Durvalumab). However, there is a still a need for potent, selective small molecule inhibitors of the PD-1/PD-L1 pathway. The approved therapies require bolus intravenous injections, are administered in high dose and have a long half life. The long residence time of these mAbs could contribute to the well-documented drug-related adverse effects. Small molecule inhibitors, therefore, can provide increased oral bioavailability, increased bio-efficiency and shorted half life activity for a more controllable treatment, particular in the case of auto-immune or other adverse effects. Rational and structure guided de novo design approaches were used to design novel small molecule PD-1/PD-L1 pathway inhibitors; potency of these inhibitors was assessed in an in-vitro TR-FRET assay. Checkpoints signaling reporter assays as well as ex-vivo co-culture assays were used to assess the ability of the compounds to restore T-cell proliferation and function. Three novel chemical series as potent PD-1/PD-L1 pathway inhibitors are being developed for the treatment of cancer. Compounds from these series showed strong in vitro potency of 0.01 to 0.2 µM against PD-1/PD-L1. JBI-426 exhibited an IC50 of 0.04 µM and no cytotoxicity against cancer cell proliferation per se. JBI-426 showed good in vitro ADME properties in terms of aqueous solubility, metabolic stability, permeability and excellent oral bioavailability in mouse pharmacokinetics. In a RENCA syngeneic model, oral administration of JBI-426 at 50 mg/kg resulted in a strong tumor growth inhibition, comparable (or better) than the PD-L1 mAb, and was well tolerated. The effect of JBI-426 on tumor infiltrating lymphocytes was also assessed; a significant increase in CD8+ cytotoxic lymphocytes was observed. Further studies to assess additional compounds from the three chemical series are underway. The oral administration route of these PD-1/PD-L1 inhibitors would provide an attractive alternate to the currently available antibodies in treating cancer either as a stand-alone therapy or in combination with other immuno-modulatory agents, as well as other standard of care agents. Citation Format: Sivanandhan Dhanalakshmi, Shivani Garapaty, Chandregowda Venkateshappa, Guru Pavan Seerapu, Reshma Das, Pradeep Nagaraj, Ronodip Kar, Anuj Kumar Singh, Venkatesha Ashokkumar Venkatesubbiah, Ramakishore VP Putta, Muralidhar Pendyala, Girisha Lokesh, Hari Madaka, Harikrishna Reddy Thummuru, Shikas AP, Prateeksha Anchan, Prathima Bhat, Rudresha G, Mohd Zainuddin, Krishnakumar V, Ramachandraiah Gosu, Rajendra Kristam, Jeyaraj DA, Sriram Rajagopal. Novel, heterocyclic small molecule inhibitors of PD-1 and PD-L1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5552.

Introduction: Arginine methylation deregulation in cancer has been well studied and PRMT5 which modulates dimethylation of arginine has emerged as an attractive therapeutic strategy in various cancer types, including lung cancer, lymphoma, glioblastoma, pancreatic cancers, etc. PRMT5 is over-expressed in multiple cancers leading to repression of tumor suppressor genes and genetic studies have identified it is a validated target in lymphoma. Recently, dysregulation of the splicing machinery in cancers has been identified to be one of the therapeutic vulnerabilities for PRMT5 inhibition, especially in glioblastoma. Therefore, inhibitors selectively targeting PRMT5 could be of high clinical value, especially in cancers with defects in spliceosome machinery. Methods: Rational design and structure based drug design were used to identify novel PRMT5 inhibitors. To assess in vitro potency, flash plate based activity assay was used. Cell based activity of these inhibitors was assessed by measuring the symmetrical dimethylation of known cellular protein SmD3 by ELISA and Western blotting. Long term cell proliferation assays were used to assess the functional effect of PRMT5 inhibition. Tumor growth inhibition was measured in orthotopic glioblastoma model in mice. Results: One of the lead PRMT5 inhibitors had an in vitro potency of 3 nM in the biochemical assay which translated well in the cell based SDMA ELISA where the EC50 was <10 nM. Anti-proliferative activity of this molecule in lymphoma, leukaemia, SCLC, pancreatic, lung and glioblastoma cell lines ranged from ~16 nM to 1035 nM. This molecule showed acceptable in vitro ADME properties in terms of aqueous solubility and metabolic stability and excellent oral bioavailability in rodent pharmacokinetics. In Z-138 mantle cell lymphoma xenograft model, oral administration of the lead compound at 50 mg/kg resulted in strong and complete (~95 %) tumour growth inhibition and with a concomitant complete inhibition of SDMA. The lead compound was well tolerated with no reduction in body weight at the tested doses. Interestingly, this molecule showed excellent brain exposure sufficient to achieve target engagement for 10h and significant tumour growth inhibition of orthotopic brain tumors by oral dosing. Repeat dose non-GLP study in rodents clearly demonstrated the safety of this molecule. Conclusion: Given the therapeutic importance of PRMT5 in glioblastoma and other lymphomas, this molecule will be extremely valuable in treating these cancers both as a standalone therapy and in combination with other standard of care agents. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Naveen Sadhu M, Chandru Gajendran, Saravanan Vadivelu, Natarajan Tamizharasan, Indu N. Swamy, Santhosh Viswakarma, Amir Siddiqui, Saif wahid, Mohammed Zainuddin, Rudresh G, Prashanthi Daram, Ramchandraiah Gosu, Dinesh Tiagaraj, Shivani Garapaty, Sreekala Nair, Namratha Kapoor. Selective, novel, small molecule PRMT5 inhibitors for treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1128.