Allosteric inhibition of HTRA1 activity by a conformational lock mechanism to treat age-related macular degeneration

Gerhardy, Stefan; Ultsch, Mark; Tang, Wanjian; Green, Evan; Holden, Jeffrey K.; Li, Wei; Estevez, Alberto; Arthur, Christopher J.; Tom, Irene; Rohou, Alexis; Kirchhofer, Daniel

doi:10.1038/s41467-022-32760-9

Cited by 6 publications

(17 citation statements)

References 86 publications

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“…4D, inset); thus, we speculate that the binding of FOLR3 to this region might inhibit catalysis by preventing substrate access to the active site. The model of FOLR3 also makes direct contacts with “LoopA” of HTRA1: An antibody binding solely to this loop has been shown to allosterically inhibit HTRA1 ( 38 ), thus offering a secondary means by which binding of FOLR3 at this location may inhibit HTRA1.…”

Section: Resultsmentioning

confidence: 99%

Secreted folate receptor γ drives fibrogenesis in metabolic dysfunction–associated steatohepatitis by amplifying TGFβ signaling in hepatic stellate cells

Quinn,

Rico,

Merali

et al. 2023

Sci. Transl. Med.

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Hepatic fibrosis is the primary determinant of mortality in patients with metabolic dysfunction–associated steatohepatitis (MASH). Transforming growth factor–β (TGFβ), a master profibrogenic cytokine, is a promising therapeutic target that has not yet been translated into an effective therapy in part because of liabilities associated with systemic TGFβ antagonism. We have identified that soluble folate receptor γ (FOLR3), which is expressed in humans but not in rodents, is a secreted protein that is elevated in the livers of patients with MASH but not in those with metabolic dysfunction–associated steatotic liver disease, those with type II diabetes, or healthy individuals. Global proteomics showed that FOLR3 was the most highly significant MASH-specific protein and was positively correlated with increasing fibrosis stage, consistent with stimulation of activated hepatic stellate cells (HSCs), which are the key fibrogenic cells in the liver. Exposure of HSCs to exogenous FOLR3 led to elevated extracellular matrix (ECM) protein production, an effect synergistically potentiated by TGFβ1. We found that FOLR3 interacts with the serine protease HTRA1, a known regulator of TGFBR, and activates TGFβ signaling. Administration of human FOLR3 to mice induced severe bridging fibrosis and an ECM pattern resembling human MASH. Our study thus uncovers a role of FOLR3 in enhancing fibrosis.

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Section: Resultsmentioning

confidence: 99%

Secreted folate receptor γ drives fibrogenesis in metabolic dysfunction–associated steatohepatitis by amplifying TGFβ signaling in hepatic stellate cells

Quinn,

Rico,

Merali

et al. 2023

Sci. Transl. Med.

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“…2-4 cm À 1 ) from those of the pristine 5 e and C 60 . [34] The most intense Raman lines corresponding to the breathing mode A g (1) and pentagonal pinch mode A g (2) of C 60 were also shifted from 496 and 1462 cm À 1 to 494 and 1468 cm À 1 , respectively. The other Raman-active modes of H g symmetry of C 60 also shifted as follows: 428 !…”

Section: Methodsmentioning

confidence: 95%

“…To elucidate the pure effect of C-alkyl substituents in conformational rigidification, we synthesized and characterized hydrogen-bonding-free OMRs 4 (Scheme 1 and Pages S4-S6). [4a, 8d, 23] The variable low-temperature (up to À 90 °C) 1 Can the α-branched C-alkyl substituents induce conformation locking in the hydrogen-bonding-free arm-extended m-cyclophanes as well? To answer this, we synthesized dissymmetric α-branched CÀ iPr 5 e and CÀ Cy 5 f coumarin- [4]arenes (CAs as racemates up to 93 % yield, Scheme 1 and Pages S6-S7) from the corresponding TMRs 2 e-f. We followed our optimized two-step method developed for 5 b-d. [9] Delightfully, the 1 H NMR signals of CAs 5 e and 5 f did not split up to À 90 °C (Figures 2B and S30) attesting to their conformational rigidity.…”

Section: Synthesis Of M-cyclophane Macrocycles and Solution-state Cha...mentioning

confidence: 99%

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Leveraging Torsional and Steric Strains: A Pre‐macrocyclization Strategy Enables Conformation‐Specific Fullerene Binding in m‐Cyclophanes

Kumar,

Mani Kandan,

Balakrishnan

et al. 2023

Angew Chem Int Ed

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Though the chemistry of resorcinarenes is half a century old, the conformationally‐locked resorcinarene crowns are generally constructed using hydrogen bonds or covalent tethers. Often, covalent tethering involves extra post‐macrocyclization steps involving upper‐rim functionalities. We have leveraged the torsional and steric strains through α‐substituents of the lower‐rim C‐alkyl chains and accomplished conformationally‐rigid fluorescent m‐cyclophane deep‐crowns in a predetermined way. The strategy offers a pre‐macrocyclization route conserving upper‐rim functionalities, an aspect overlooked in resorcinarene chemistry. X‐ray structural and computational analyses unveil the cause for conformational rigidity in m‐cyclophanes due to α‐branching in C‐alkyls (linear vs. α‐/β‐branched). The conformationally‐locked fluorescent deep‐crown with a preorganized cavity captures hydrophobic spherical guest C60 in both solution and solid states specifically, when compared to conformationally‐dynamic boats, enabling conformation‐specific binding.

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“…HTRA1 forms a homotrimer through its protease domains that positions the three active sites in close proximity such that antibodies are unable to simultaneously access them. Indeed, known inhibitory antibodies of HTRA1 bind to epitopes peripheral to the active site and use allosteric mechanisms 46 , 47 . In contrast, small compact peptides such as CKPs do not have these spatial restrictions, which increases the available surface area for functional binding, including the occupation of all three active sites within an HTRA1 trimer.…”

Section: Introductionmentioning

confidence: 99%

Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region

Li,

Wei,

Ultsch

et al. 2024

Nat Commun

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Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1’ site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.

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Allosteric inhibition of HTRA1 activity by a conformational lock mechanism to treat age-related macular degeneration

Cited by 6 publications

References 86 publications

Secreted folate receptor γ drives fibrogenesis in metabolic dysfunction–associated steatohepatitis by amplifying TGFβ signaling in hepatic stellate cells

Secreted folate receptor γ drives fibrogenesis in metabolic dysfunction–associated steatohepatitis by amplifying TGFβ signaling in hepatic stellate cells

Leveraging Torsional and Steric Strains: A Pre‐macrocyclization Strategy Enables Conformation‐Specific Fullerene Binding in m‐Cyclophanes

Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region

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