The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins.

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.

Impact of COVID-19 on Work Loss in the United States- A Retrospective Database Analysis.

ObjectivesThis study investigates the utilization of work absence benefits among United States (US) employees diagnosed with COVID-19, examining frequency, duration, cost, and types of work loss benefits used.MethodsThis retrospective analysis of the Workpartners Research Reference Database (RRDb) included employees eligible for short- and long-term disability (STD and LTD employer-sponsored benefits, respectively), and other paid work absence benefits from 2018-2022. Workpartners RRDb includes over 3.5 million employees from over 500 self-insured employers across the US. Employees were identified by codes from adjudicated medical and disability claims for COVID-19 (2020-2022) and influenza, as well as prescription claims for COVID-19. Associated payments were quantified for each absence reason.ResultsApproximately 1 million employees were eligible for employer-sponsored paid leave benefits between January 2018 and December 2022. The mean age was 37 years (22% >50 years), and 49.9% were female. COVID-19 was the 2nd most common reason for an STD claim (6.9%) and 13th for an LTD claim (2020-2022). The mean duration for COVID-19 STD claims was 24 days (N = 3731, mean claim=$3477) versus 10 days for influenza (N = 283, mean claim=$1721). The mean duration for an LTD claim for COVID-19 was 153 days (N = 24, mean claim=$19,254). Only 21.5% of employees with STD claims in the COVID-19 cohort had prior COVID-19-associated medical or pharmacy claims; over half (range 53%-61%) had documented high risk factors for severe COVID-19.ConclusionCOVID-19 and influenza have the potential to cause work loss in otherwise healthy employees. In this analysis, COVID-19 was the second most frequent reason for an STD claim at the start of the pandemic and remained high (ranked 5th) in 2022. These results highlight the impact of COVID-19 on work loss beyond the acute phase. Comprehensively evaluating work loss implications may help employers prioritize strategies, such as vaccinations and timely treatments, to mitigate the impact of COVID-19 on employees and their companies.

COVID-19 results in short- and long-term symptoms that may affect employees' ability to work. Short- and long-term disability (STD and LTD, respectively), other work absences, and medical and pharmacy claims from the Workpartners Research Reference Database were analyzed for US adult (≥18 years) employees. COVID-19 claims were identified using the Center for Disease Control and Prevention recommended International Classification of Diseases codes during the analysis from 2020 to 2022. During 2020 to 2022, COVID-19 ranked as the second most frequent reason for STD claims and 13^th most frequent among LTD claims. Influenza ranked 58^th overall with no LTD claims (2018­2022). The average COVID-19 STD claim lasted 24 days and cost employers $3477 per claim, and LTD claims averaged 153 days, costing $19,254. Only 21.5% of employees with STD claims in the COVID-19 cohort had prior COVID-19-associated medical or pharmacy claims, and over half (range 53%-61%) had a documented high-risk factor for severe COVID-19. Our results highlight the ongoing and substantial impact of COVID-19 on work absence benefit utilization beyond the acute phase. This analysis demonstrates the need for employers and researchers to review all available medical, pharmacy, and disability claims to assess the acute and long-term impact of COVID-19 on employees and prioritize mitigation strategies to reduce the burden of the virus to their employees.

Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

The N-terminal length and side-chain composition of CXCL13 affect crystallization, structure and functional activity.

CXCL13 is the cognate chemokine agonist of CXCR5, a class A G-protein-coupled receptor (GPCR) that is essential for proper humoral immune responses. Using a `methionine scanning' mutagenesis method on the N-terminus of CXCL13, which is the chemokine signaling region, it was shown that minor length alterations and side-chain substitutions still result in CXCR5 activation. This observation indicates that the orthosteric pocket of CXCR5 can tolerate these changes without severely affecting the activity. The introduction of bulk on the ligand was well tolerated by the receptor, whereas a loss of contacts was less tolerated. Furthermore, two crystal structures of CXCL13 mutants were solved, both of which represent the first uncomplexed structures of the human protein. These structures were stabilized by unique interactions formed by the N-termini of the ligands, indicating that CXCL13 exhibits substantial N-terminal flexibility while the chemokine core domain remains largely unchanged. Additionally, it was observed that CXCL13 harbors a large degree of flexibility in the C-terminal extension of the ligand. Comparisons with other published structures of human and murine CXCL13 validate the relative rigidity of the core domain as well as the N- and C-terminal mobilities. Collectively, these mutants and their structures provide the field with additional insights into how CXCL13 interacts with CXCR5.

Characterization, Dynamics, and Mechanism of CXCR4 Antagonists on a Constitutively Active Mutant.

The G protein-coupled receptor (GPCR) CXCR4 is a co-receptor for HIV and is involved in cancers and autoimmune diseases. We characterized five purine or quinazoline core polyamine pharmacophores used for targeting CXCR4 dysregulation in diseases. All were neutral antagonists for wild-type CXCR4 and two were biased antagonists with effects on ß-arrestin-2 only at high concentrations. These compounds displayed various activities for a constitutively active mutant (CAM). We use the IT1t-CXCR4 crystal structure and molecular dynamics (MD) simulations to develop two hypotheses for the activation of the N1193.35A CAM. The N1193.35A mutation facilitates increased coupling of TM helices III and VI. IT1t deactivates the CAM by disrupting the coupling between TM helices III and VI, mediated primarily by residue F872.53. Mutants of F872.53 in N1193.35A CXCR4 precluded constitutive signaling and prevented inverse agonism. This work characterizes CXCR4 ligands and provides a mechanism for N1193.35A constitutive activation.

An Analysis of MIF Structural Features that Control Functional Activation of CD74.

For more than 15 years, the tautomerase active site of macrophage migration inhibitory factor (MIF) and its catalytic residue Pro1 have been being targeted for the development of therapeutics that block activation of its cell surface receptor, CD74. Neither the biological role of the MIF catalytic site nor the mechanistic details of CD74 activation are well understood. The inherently unstable structure of CD74 remains the biggest obstacle in structural studies with MIF for understanding the basis of CD74 activation. Using a novel approach, we elucidate the mechanistic details that control activation of CD74 by MIF surface residues and identify structural parameters of inhibitors that reduce CD74 biological activation. We also find that N-terminal mutants located deep in the catalytic site affect surface residues immediately outside the catalytic site, which are responsible for reduction of CD74 activation.