Biology subjects

Vashisth, H.

Publications and source records attributed to Vashisth, H..

6 recordsLinked to original sources

bioRxiv2026

Molecular Determinants of Allosteric Inhibitor Affinity and Selectivity in PDE5

Allosteric inhibitors targeting unique regulatory pockets provide an opportunity for selective modulation of proteins within the phosphodiesterase (PDE) family. In this work, we investigate the molecular determinants for binding of an evodiamine derivative (EVO) to PDE5 and the structural basis for differential recognition of this inhibitor in PDE6 isoforms. We integrated structural modeling, all-atom molecular dynamics (MD) simulations, and extensive alchemical free-energy calculations to quantify residue-level contributions to inhibitor binding in PDE5 through systematic alanine scanning and PDE6-derived mutations. Thermodynamic analysis based on free energy identified key residues that govern EVO binding through a network of hydrogen bonds and hydrophobic contacts. Specifically, we examined structural changes associated with allosteric pocket variations in PDE5, identified key structural elements, and evaluated the role of chemical substituents of EVO in allosteric inhibition. Moreover, PDE6-derived substitutions revealed isoform specific effects, with PDE6-rod substitutions resulting in stronger destabilization than PDE6-cone substitutions. Together, these results define the energetic and structural basis for allosteric inhibition of PDE5 and provide mechanistic insights for the rational design of selective PDE inhibitors. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712116v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@5934b4org.highwire.dtl.DTLVardef@9346d7org.highwire.dtl.DTLVardef@10fcd8corg.highwire.dtl.DTLVardef@7fc5c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Molecular Interactions of Viral Insulin/IGF-like Peptides with Zebrafish Receptors

Signaling through the insulin receptor (IR) and the type 1 insulin-like growth factor receptor (IGF1R) is modulated by secreted hormones and growth factor ligands (e.g. insulin and insulin-like growth factor 1, IGF1). Impaired signaling in these receptors often leads to diabetes and oncogenic diseases. The discovery of entirely novel viral insulin/IGF-like peptides (VILPs) that can stimulate receptors from the insulin family has raised questions about their structures and binding modes to receptors. These peptides exist in a single-chain (sc) or a double-chain (dc) configuration with folds likely similar to IGF1 and insulin, respectively. The interactions of VILPs with the human receptors are beginning to be mapped but little is known about their interactions with the receptors in fish-the host organism for viruses known to carry these peptide sequences. We have previously reported [Chuard et al., Cell Rep. 2025 44(8):116149] structural models of several VILPs from the Iridoviridae virus family bound to their cognate receptors in Zebrafish (Zeb). In this work, we conducted all-atom molecular dynamics (MD) simulations of these peptides and their receptor-bound complexes along with free energy calculations to assess the energetic contributions of VILP residues for their binding to Zebrafish receptors. Most of the observed Zeb insulin/Zeb {micro}IR and Zeb IGF1/Zeb {micro}IGF1R site 1 interactions are consistent with previously known interactions of human peptides with their receptors, highlighting similarities in their binding modes. However, we also report some non-conserved residues in VILPs that establish significant and unique interactions with residues in Zeb receptors. Furthermore, we identified residues in each VILP which can be potentially mutated into conserved insulin/IGF1 residues to possibly enhance the binding affinity of these peptides.

biophysics↗

bioRxiv2025

Conformational Dynamics in Insulin Receptor Kinase Reveals a Type III Allosteric Pocket

Allosteric modulation of kinases is a promising approach for pharmacological intervention, particularly for designing selective kinase modulators. However, the structural information on allosteric pockets remains limited for most kinases in the human kinome. In this work, we present comprehensive characterization of a type III allosteric pocket in the insulin receptor kinase (IRK) by uncovering its structural features and conformational dynamics. Specifically, we used microsecond-scale atomistic molecular dynamics (MD) simulations to investigate apo and inhibitor-bound IRK structures. Our findings suggest that the type III allosteric site is a "back pocket" in IRK, sandwiched between the N-terminal and the C-terminal lobes, beneath the C-helix and the {beta} - sheets of the N-terminal lobe. It has a hydrophobic cleft composed of both aliphatic and aromatic non-polar residues and a charge center to facilitate electrostatic interactions. We explored the binding of an experimentally known IRK inhibitor to the newly discovered allosteric pocket. Our results indicate that the C-helix adopts an "out" conformation stabilized by the inhibitor which promotes an inactive conformation of the kinase. Furthermore, we observed a helical intermediate formation in the activation loop and a stable "DFG-out" conformation in both inhibitor-bound and unbound states. Our results also suggest that the residue M1051 in IRK functions as a gatekeeper residue, essential for maintaining the structural integrity of the C-helix and regulating the binding of the allosteric inhibitor. Our findings are relevant for developing allosteric IRK modulators and informing therapeutic strategies targeting proteins in the insulin receptor family.

biophysics↗

bioRxiv2024

Viral Insulin/IGF-like Peptides Selectively Activate Host Insulin/IGF Signaling pathways during Grouper Iridovirus Infection

Until our recent discovery of viral insulin/IGF-1-like peptides (VILPs), examples of viral mimicry were mostly limited to immunomodulatory proteins and growth factors. We previously showed that six viruses in the Iridoviridae family encode genes mimicking insulin and IGF-1. While VILP-carrying viruses are isolated from fish, the role of VILPs in host-pathogen interactions remain elusive. In this study, we used the Grouper Iridovirus (GIV), one of the VILP-carrying viruses, to examine the impact of the GIV-VILPs on the host IGF-1R/IR system during infection. Our structural analysis revealed that GIV-VILP is 35% identical to both zebrafish insulin and 35 IGF-1. We also showed that GIV-VILPs are early viral transcripts and resulting peptides are secreted during viral infection. Both single-chain (sc, resembling IGF-1) and double chain (dc, resembling insulin) forms of chemically synthesized GIV-VILPs can stimulate insulin receptor (IR) and IGF-1 receptor (IGF1R) phosphorylation. They could also stimulatepost-receptor signaling in both grouper kidney (GK) and zebrafish AB.9 cells as potently as insulin. Notably, supernatants obtained from GIV-infected cells could stimulate insulin/IGF system in a dose dependent manner. Interestingly, GIV-VILP was able to selectively activate the Akt/PI3K pathway while had no or minimal effect on Erk/MAPK pathway. Using either IR, IGF1R or dual inhibitors, we illustrated that inhibition of IR signaling suppressed GIV viral replication while inhibition of IGF1R enhanced viral replication. Consistent with selective signaling action of PI3K/Akt pathway, inhibition of the Akt reduced GIV viral replication however Erk pathway inhibition did not affect it. In summary, GIV-VILP is produced during GIV infection and manipulates host insulin/IGF signaling in a selective manner. Our findings reveal a previously unknown viral strategy in which viruses mimic a host hormone to exploit the hosts endocrine system. This discovery unveils a novel viral pathogenesis mechanism, broadens our understanding of viral mimicry, and opens a new avenue to better understand viral strategies to manipulate the host.

microbiology↗

bioRxiv2024

Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt {Lambda}-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having physiologically representative lipid composition has not been observed. We address this limitation by employing coarsegrained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/593448v3_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@107bfdforg.highwire.dtl.DTLVardef@1c5ca00org.highwire.dtl.DTLVardef@50d859org.highwire.dtl.DTLVardef@1abc266_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Structural Models for a Series of Allosteric Inhibitors of IGF1R Kinase

The allosteric inhibition of Insulin-like Growth Factor Receptor 1 Kinase (IGF1RK) is a potential strategy to overcome selectivity barriers in targeting receptor tyrosine kinases. We constructed structural models of a series of 12 indole-butyl-amine derivatives which have been reported as allosteric inhibitors of IGF1RK. We further studied dynamics and interactions of each inhibitor in the allosteric pocket via all-atom explicit-solvent molecular dynamics (MD) simulations. We discovered that a bulky carbonyl substitution at the R1 indole ring is structurally unfavorable for inhibitor binding in the IGF1RK allosteric pocket. Moreover, we found that the most potent derivative (termed C11) acquires a distinct conformation, forming an allosteric pocket channel with better shape complementarity and interactions with the receptor. In addition to a hydrogen bonding interaction with V1063, the cyano derivative C11 forms a stable hydrogen bond with M1156, which is responsible for its unique binding conformation in the allosteric pocket. Our findings show that the position of chemical substituents at the R1 indole ring with different pharmacophore features influences molecular interactions and binding conformations of the indole-butyl-amine derivatives, hence dramatically affecting their potencies. Our results provide a structural framework for the design of allosteric inhibitors with improved affinities and specificities against IGF1RK.

biophysics↗