Inhibitor of RNA polymerase ii

Abstract

An inhibitor of RNA polymerase II is described, wherein said inhibitor is selected a moiety which targets a protein selected from cyclin kinase 12 (CDK12) or its recruiting protein PAF1C. Particular examples of such inhibitors are polypeptides expressed by a gene selected from lldD, lldR, nlpD or rfaH of a bacterial species, such as a commensal bacteria or asymptomatic carrier, or a variant of said protein. Inhibitors may be based upon bacterial Sigma S or NplD proteins. These inhibitors are useful in therapies, to suppress protein expression. Thus they may be used as immunosuppressants, anti-inflammatory or anti-infection agents.

Description

[0001]The present invention relates to factors and moieties that can modulate host protein expression, for example, a factor having immunosuppressant activity, to methods of preparing the factors and moieties and to their use in therapy.BACKGROUND TO THE INVENTION[0002]The multi-subunit RNA polymerase II complex (Pol II) is essential for protein expression in eukaryotes. Transcription cycle has been extensively characterized to show that around 10% of all expressed genes are involved in transcriptional regulation, assembly and control of the Pol II complex. In eukaryotic cells, RNA polymerase II catalyzes the synthesis of mRNA and small nuclear RNA. Pol II transcription cycle consists of several stages, termed preinitiation, initiation, promoter clearance, during which Pol II pauses at the promoter proximal site, followed by escape from pausing, productive elongation and termination.[0003]For specificity, the assembly of Pol II is tightly controlled and the efficiency of transcripti...

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Benefits of technology

[0038]As described herein, two, closely related genes have been identified in the WT strain E. coli 83972, based on a screen for “loss of function” mutants. A Tyr209Hi amino acid change in NlpD, abolished the inhibitory effect on Pol II phosphorylation. NlpD is a secreted protein with a 25 aa-signal sequence and potential signal peptidase cleavage site and is transported to the bacterial periplasm. NlpD together with other LytM (lysostaphin)—domain-containing factors is required for septal proteoglycan splitting and daughter cell separation. When overexpressed, nlpD changes bacterial morphology, due to the activation of the cell wall hydrolase AmiC. The mutation in SN25 appeared to be located within the AmiC binding site, suggesting that this mutant has lost the ability to activate AmiC and thus to facilitate the secretion of bacterial components and their interactions with the host cell. The rapid reduction in PAF1C and CDK12 protein levels suggested that host proteases or ubiquitinases might be activated. Uehara et al., 2010 discuss four possible mechanisms of bacterial amidase activation by LytM factors including NlpD. These include 1) allosteric or 2) covalent modification (e.g. proteolytic processing) of amidases by the LytM factors, 3) facilitated substrate association of the amidases and 4) prior deformation or hydrolysis of bonds in the PG substrate. Therefore, similarly to activation of the amidase in bacterial cells, NlpD might activate some other enzymes in the host cells.

[0041]NlpD also exerts its effect through transcriptional control of rpoS and rpoS-dependent genes. The transcription of rpoS is regulated from a common nlpD promoter and from additional sites within the nlpD ORF. Sigma S is an important regulator of more than 20 stationary phase genes and operons such as genes required for multiple-stress resistance. In addition, as NlpD and Sigma S proteins are encoded by the same gene cluster and are derived from the same polycystronic RNA, direct interactions cannot be excluded. It can be hypothesized that NlpD allosterically modifies Sigma S, facilitating its transport and rendering it active in binding and melting of host cell DNA.

[0043]Without being bound by theory, it is possible that disruption of the pre-initiation complex by Sigma S might be a key step, affecting downstream transcriptional activity. The applicants have shown that bacterial RpoS competes with human TBP for binding to TATA box DNA. By dislodging TBP from its binding site, RpoS may thus prevent pre-initiation complex formation and therefore the binding of Pol II to specific promoters. A general effect on gene expression is supported by a clinical study, showing reduced expression of >60% of all genes in circulating blood cells in patients inoculated with E. coli 83972. Despite the inhibition of a large number of genes, effects were much less pronounced than when a pharmacological inhibitor was used, however, suggesting specificity. In eukaryotic promoters, PIC formation and binding of general transcription factor II B (TFIIB) recruits Pol II to the promoter. II B (TFIIB) consists of 4 functional domains—N-terminal Zn ribbon, B reader, B linker and Core domain. Core domain in eukaryotes is required to stabilize the TBP-DNA complex and the Zn ribbon recruits RNA Pol II.

Problems solved by technology

However, as a broad spectrum suppressor of protein expression, Pol-II inhibitors may find application in any therapy where host protein is over-expressed or problematic.

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