A cell-based high-throughput screen identifies drugs that cause bleeding disorders by off-targeting the vitamin K cycle

Abstract

Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding with obvious reasons are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that impact the biosynthesis of active vitamin K-dependent (VKD) coagulation factors with possible off-target effects. The NIH Clinical Collection (NCC) library containing 727 drugs was screened and 9 drugs, including the most commonly prescribed anticoagulant warfarin, were identified. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), while idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other three drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs targeting VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.

Insights into vitamin K-dependent carboxylation: home field advantage

“…In fact, this group recently used this assay in a high-throughput capacity to screen small molecules that impact the vitamin K cycle, an exercise that would be not possible using prior approaches. 16 In summary, this elegant report confirms the critical role that the propeptide region plays in carboxylation of VKD proteins and highlights the utility of a novel cell-based assay that enables researchers to study membrane-associated enzymes in their natural, home environment.…”

A novel vitamin K derived anticoagulant tolerant to genetic variations of vitamin K epoxide reductase

“…We have shown previously that this background activity is a result of residual K vitamins in the fetal bovine serum supplemented in the cell culture medium. 40 Therefore, these results indicate that VK-M-COT could be an inhibitor for VKD carboxylation.…”

“…To confirm that the vitamin K derivatives unable to support VKD carboxylation in the UBIAD1‐knockout reporter cells was due to the absence of the UBIAD1 gene, the UBIAD1 cDNA was reintroduced back into these reporter cells by transient expression and the reporter‐protein carboxylation in the transfected cells was determined. To confirm that the vitamin K analogue supported VKD carboxylation as a result of the conversion of the vitamin K derivatives to MK‐4 by UBIAD1, HEK293, or UBIAD1‐knockout, HEK293 reporter cells were cultured with the vitamin K derivatives and the production of MK‐4 within these cells were determined using an HPLC assay 40 …”

“…To confirm that the vitamin K analogue supported VKD carboxylation as a result of the conversion of the vitamin K derivatives to MK-4 by UBIAD1, HEK293, or UBIAD1-knockout, HEK293 reporter cells were cultured with the vitamin K derivatives and the production of MK-4 within these cells were determined using an HPLC assay. 40…”

“…Next, we determined KO and vitamin K reductase activities in HEK293 cells by directly measuring K vitamins within the cells using a conventional reversed-phase HPLC assay. 40 is unstable and difficult to accurately quantify from cells directly, 41 we coupled the vitamin K reduction and epoxidation reactions to measure the final stable product KO ( Figure 2C). Our results show that VK-M-COT significantly decreased both the reduction of KO ( Figure 3A) and vitamin K ( Figure 3B), which are consistent with the results from our cell-based carboxylation activity assays ( Figure 2D).…”