Mechanism of action of hepatitis B virus S antigen transport-inhibiting oligonucleotide polymer, STOPS, molecules

Abstract

A functional cure of chronic hepatitis B requires eliminating the hepatitis B virus (HBV)-encoded surface antigen (HBsAg), which can suppress immune responses. STOPS are phosphorothioated single-stranded oligonucleotides containing novel chemistries that significantly reduce HBsAgs produced by HBV-infected liver cells. The STOPS molecule ALG-10000 functions inside cells to reduce the levels of multiple HBV-encoded molecules. However, it does not bind HBV molecules. An affinity resin coupled with ALG-10000 was found to bind several proteins from liver cells harboring replicating HBV. Silencing RNAs targeting host factors SRSF1, HNRNPA2B1, GRP78 (HspA5), RPLP1, and RPLP2 reduced HBsAg levels and other HBV molecules that are concomitantly reduced by STOPS. Host proteins RPLP1/RPLP2 and GRP78 function in the translation of membrane proteins, protein folding, and degradation. ALG-10000 and the knockdowns of RPLP1/2 and GRP78 decreased the levels of HBsAg and increased their ubiquitination and proteasome degradation. GRP78, RPLP1, and RPLP2 affected HBsAg production only when HBsAg was expressed with HBV regulatory sequences, suggesting that HBV has evolved to engage with these STOPS-interacting molecules. The STOPS inhibition of HBsAg levels in HBV-infected cells occurs by sequestering cellular proteins needed for proper expression and folding of HBsAg.

Hepatitis B virus movement through the hepatocyte: An update

“…Recently, a new class of NAPs called S‐antigen transport‐inhibiting oligonucleotide polymers (STOPS) were developed that are able to inhibit HBV infection and HBsAg production in vitro. Again, they do not bind HBV molecules, but rather targets host factors, including the BiP chaperone, RPLP1, and RPLP2 (60S acidic ribosomal protein P1 and P2), that are required for the proper translation and folding of S/sSVPs (Kao et al., 2022).…”

Editorial: In vitro mechanistic evaluation of nucleic acid polymers: A cautionary tale

“…Recently published in vitro data 5 suggest that NAPs modified with locked nucleic acids (LNAs) have substantially improved potency compared with previously published NAPs and suggested mechanisms of action and potential host targets very different from those previously published for NAPs. The even more recent announcement of the abandonment of the LNA-modified NAP ALG-10133 and the entire LNA-modified NAP platform because of a lack of antiviral activity in humans 6 raises serious questions regarding these recently published in vitro data.…”

“…These artifacts prevent the correct comparison of relative antiviral activity between NAPs with different modifications and result in the observation of many inhibitory effects in vitro that do not occur in vivo or in humans. The disconnect between transfection data presented with NAPs in vitro 5 , 31 , 36 and in humans 3 , 6 , 20 is a clear validation of these artifacts. Future evaluations of NAPs in vitro must use suitable systems as described above, which have been validated with known control NAPs, 7 , 33 and target identification must be performed under well-controlled conditions.…”

“… 34 , 35 As such, in ALG-10133, the doping of the uniform 2′O-methylation present in REP 2165 ( Figure 1 ) substantially reduced the hydration of this NAP and allowed it to be efficiently transfected, yielding apparent (but artifactually) increased antiviral activity by transfection relative to REP 2139, 31 similar to ALG-10000. 5 The second reason for the failure of ALG-10133 is the structural rigidity LNA imparts to the NAP. The unstructured and flexible nature of NAPs is essential for their optimal antiviral effect 1 , 2 , 7 because this flexibility allows optimal interaction with uncomplexed amphipathic alpha helices, for example in the J-domain of DNAJB12 ( Figure 5 ).…”

RNA interference as a novel treatment strategy for chronic hepatitis B infection

“…Multiple novel therapeutics, broadly classified as virus-targeting agents and immunomodulators, are being actively investigated as strategies for achieving functional cure [ 5 ]. Virus-targeting agents inhibit different steps of the HBV lifecycle and agents in development include: 1) entry inhibitors: which inhibit HBV docking and hepatocyte entry [ 6 ]; 2) polymerase inhibitors: which inhibit HBV polymerase competitively (conventional NAs) or non-competitively (novel active site polymerase inhibitor nucleotide) [ 7 ]; 3) RNA silencers: which inhibit HBV messenger RNA (mRNA) translation and viral protein production [ 8 ]; 4) capsid assembly modulators: which inhibit HBV nucleocapsid assembly and pregenomic RNA encapsidation [ 9 ]; 5) viral protein export inhibitors: which inhibit HBV antigen release from hepatocytes [ 10 , 11 ]; and 6) farnesoid X receptor (FXR) agonist: which reverses the interaction between FXR and the HBV X protein to interfere with transcription regulation [ 12 ]. Immunomodulators utilize an alternative strategy which is different from virus-targeting agents.…”