Discovery and evaluation of direct acting antivirals against hepatitis C virus

Until recently, the standard therapy for hepatitis C treatment has been interferon and ribavirin. Such treatment has only 50% efficacy and is not well tolerated. The emergence of new drugs has increased the treatment efficacy to 90%. Despite such an achievement, the success is limited since the virus mutates rapidly, causing the emergence of drug resistant forms. In addition, most new drugs were developed to treat genotype 1 infections. Thus, development of new potent antivirals is needed and drug discovery against hepatitis C is continued. In this thesis, a FRET-based protease assay was used to evaluate new pyrazinone based NS3 protease inhibitors that are structurally different to the newly approved and currently developing drugs. Several compounds in this series showed good potencies in the nanomolar range against NS3 proteases from genotype 1, 3, and the drug resistance variant R155K. We assume that these compounds can be further developed into drug candidates that possess activity against above mentioned enzyme variants. By using SPR technology, we analyzed interaction mechanisms and characteristics of allosteric inhibitors targeting NS5B polymerases from genotypes 1 and 3. The compounds exhibited different binding mechanisms and displayed a low affinity against NS5B from genotype 3. In order to evaluate the activity and inhibitors of the NS5B polymerase, we established an SPR based assay, which enables the monitoring of polymerization and its inhibition in real time. This assay can readily be implemented for the discovery of inhibitors targeting HCV. An SPR based fragment screening approach has also been established. A screen of a fragment library has been performed in order to identify novel scaffolds that can be used as a starting point for development of new allosteric inhibitors against NS5B polymerase. Selected fragments will be further elaborated to generate a new potent allosteric drug candidate. Alternative approaches have successfully been developed and implemented to the discovery of potential lead compounds targeting two important HCV drug targets

Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a

Characterization of interactions between hepatitis C virus NS5B polymerase, annexin A2 and RNA - effects on NS5B catalysis and allosteric inhibition

Background: Direct acting antivirals (DAAs) provide efficient hepatitis C virus (HCV) therapy and clearance for a majority of patients, but are not available or effective for all patients. They risk developing HCV-induced hepatocellular carcinoma (HCC), for which the mechanism remains obscure and therapy is missing. Annexin A2 (AnxA2) has been reported to co-precipitate with the non-structural (NS) HCV proteins NS5B and NS3/NS4A, indicating a role in HCC tumorigenesis and effect on DAA therapy. Methods: Surface plasmon resonance biosensor technology was used to characterize direct interactions between AnxA2 and HCV NS5B, NS3/NS4 and RNA, and the subsequent effects on catalysis and inhibition. Results: No direct interaction between AnxA2 and NS3/NS4A was detected, while AnxA2 formed a slowly dissociating, high affinity (K D = 30 nM), complex with NS5B, decreasing its catalytic activity and affinity for the allosteric inhibitor filibuvir. The RNA binding of the two proteins was independent and AnxA2 and NS5B interacted with different RNAs in ternary complexes of AnxA2:NS5B:RNA, indicating specific preferences. Conclusions: The complex interplay revealed between NS5B, AnxA2, RNA and filibuvir, suggests that AnxA2 may have an important role for the progression and treatment of HCV infections and the development of HCC, which should be considered also when designing new allosteric inhibitors

Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobody reagents

High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.We thank Dr. Junhong Yan for discussions about Nbs oligo conjugation. The Single Cell Proteomic Facility at SciLifeLab, Uppsala for supports to setup PEA assay. This work was supported by a grant (2012-5852) from the Swedish Research Council’s special call for future therapy, Swedish Research Council under grant agreement No. 2020-02258, a Torsten Söderbergs Stiftelse (M130/16) and the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ ERC Grant Agreement No. 294409, ProteinSeq and from The Swedish Collegium for Advanced Studies (SCAS).Alternative recombinant protein binders selection via proximity assay

Amino acid substitutions in human growth hormone affect secondary structure and receptor binding

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH

Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ

Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues

Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors. Helleday and colleagues describe a nanomolar MTHFD2 inhibitor that causes replication stress and DNA damage accumulation in cancer cells via thymidine depletion, demonstrating a potential therapeutic strategy in AML tumors in vivo