Structure-activity relationships of dihydropyrimidone inhibitors against native and auto-processed human neutrophil elastase.

BACKGROUND: Human neutrophil elastase (HNE) is a key driver of systemic and cardiopulmonary inflammation. Recent studies have established the existence of a pathologically active auto-processed form of HNE with reduced binding affinity against small molecule inhibitors. METHOD: AutoDock Vina v1.2.0 and Cresset Forge v10 software were used to develop a 3D-QSAR model for a series of 47 DHPI inhibitors. Molecular Dynamics (MD) simulations were carried out using AMBER v18 to study the structure and dynamics of sc (single-chain HNE) and tcHNE (two-chain HNE). MMPBSA binding free energies of the previously reported clinical candidate BAY 85-8501 and the highly active BAY-8040 were calculated with sc and tcHNE. RESULTS: The DHPI inhibitors occupy the S1 and S2 subsites of scHNE. The robust 3D-QSAR model showed acceptable predictive and descriptive capability with regression coefficient of r2 = 0.995 and cross-validation regression coefficient q2 = 0.579 for the training set. The key descriptors of shape, hydrophobics and electrostatics were mapped to the inhibitory activity. In auto-processed tcHNE, the S1 subsite undergoes widening and disruption. All the DHPI inhibitors docked with the broadened S1'-S2' subsites of tcHNE with lower AutoDock binding affinities. The MMPBSA binding free energy of BAY-8040 with tcHNE reduced in comparison with scHNE while the clinical candidate BAY 85-8501 dissociated during MD. Thus, BAY-8040 may have lower inhibitory activity against tcHNE whereas the clinical candidate BAY 85-8501 is likely to be inactive. CONCLUSION: SAR insights gained from this study will aid the future development of inhibitors active against both forms of HNE.

Exploring the role of framework mutations in enabling breadth of a cross-reactive antibody (CR3022) against the SARS-CoV-2 RBD and its variants of concern.

Cross-reactive and broadly neutralizing antibodies against surface proteins of diverse strains of rapidly evolving viral pathogens like SARS-CoV-2 can prevent infection and therefore are crucial for the development of effective universal vaccines. While antibodies typically incorporate mutations in their complementarity determining regions during affinity maturation, mutations in the framework regions have been reported as players in determining properties of broadly neutralizing antibodies against HIV and the Influenza virus. We propose an increase in the cross-reactive potential of CR3022 against the emerging SARS- CoV-2 variants of concern through enhanced conformational flexibility. In this study, we use molecular dynamics simulations, in silico mutagenesis, structural modeling, and docking to explore the role of light chain FWR mutations in CR3022, a SARS-CoV anti-spike (S)-protein antibody cross-reactive to the S-protein receptor binding domain of SARS-CoV-2. Our study shows that single substitutions in the light chain framework region of CR3022 with conserved epitopes across SARS-CoV strains allow targeting of diverse antibody epitope footprints that align with the epitopes of recently-categorized neutralizing antibody classes while enabling binding to more than one strain of SARS-CoV-2. Our study has implications for rapid and evolution-based engineering of broadly neutralizing antibodies and reaffirms the role of framework mutations in effective change of antibody orientation and conformation via improved flexibility.Communicated by Ramaswamy H. Sarma.

Chronic hM4Di-DREADD-Mediated Chemogenetic Inhibition of Forebrain Excitatory Neurons in Postnatal or Juvenile Life Does Not Alter Adult Mood-Related Behavior.

G-protein-coupled receptors (GPCRs) coupled to Gi signaling, in particular downstream of monoaminergic neurotransmission, are posited to play a key role during developmental epochs (postnatal and juvenile) in shaping the emergence of adult anxiodepressive behaviors and sensorimotor gating. To address the role of Gi signaling in these developmental windows, we used a CaMKIIα-tTA::TRE hM4Di bigenic mouse line to express the hM4Di-DREADD (designer receptor exclusively activated by designer drugs) in forebrain excitatory neurons and enhanced Gi signaling via chronic administration of the DREADD agonist, clozapine-N-oxide (CNO) in the postnatal window (postnatal days 2-14) or the juvenile window (postnatal days 28-40). We confirmed that the expression of the HA-tagged hM4Di-DREADD was restricted to CaMKIIα-positive neurons in the forebrain, and that the administration of CNO in postnatal or juvenile windows evoked inhibition in forebrain circuits of the hippocampus and cortex, as indicated by a decline in expression of the neuronal activity marker c-Fos. hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons in postnatal or juvenile life did not impact the weight profile of mouse pups, and also did not influence the normal ontogeny of sensory reflexes. Further, postnatal or juvenile hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons did not alter anxiety- or despair-like behaviors in adulthood and did not impact sensorimotor gating. Collectively, these results indicate that chemogenetic induction of Gi signaling in CaMKIIα-positive forebrain excitatory neurons in postnatal and juvenile temporal windows does not appear to impinge on the programming of anxiodepressive behaviors in adulthood.