Melcher Laboratory

AMPK, a potential target for the treatment of diabetes, obesity, and cancer

Cells use ATP to drive energy-consuming cellular processes, such as muscle contraction, cell growth, and neuronal excitation.  Adenosine-monophosphate (AMP)-activated protein kinase (AMPK) is a three-subunit protein kinase that functions as a sensor of the energy status in human cells.  Its kinase activity is triggered by energy stress (i.e., a drop in the ratio of ATP to AMP/ADP), activating ATP-generating pathways and reducing energy-consuming programs.  

To adjust energy balance, AMPK regulates:

  • Almost all cellular metabolic processes (activation of ATP-generating pathways such as glucose and fatty acid uptake and catabolism, and inhibition of energy-consuming pathways such as the synthesis of glycogen, fatty acids, cholesterol, proteins, and ribosomal RNA)
  • Whole-body energy balance (appetite regulation in the hypothalamus via leptin, adiponectin, ghrelin, and cannabinoids) 
  • Many nonmetabolic processes (cell growth and proliferation, mitochondrial homeostasis, autophagy, aging, neuronal activity, and cell polarity).  

Due to its central roles in the uptake and metabolism of glucose and fatty acids, AMPK is an important pharmacological target for the treatment of diabetes and obesity.  Moreover, AMPK activation restrains the growth and metabolism of tumor cells and has thus become an exciting new target for cancer therapy.  In this project we strive to determine the structural mechanisms of AMPK regulation by direct binding of AMP, ADP, ATP, drugs, and glycogen, in order to provide a structural framework for the rational design of new therapeutic AMPK modulators. 

Signaling by ABA, a central regulator of the plant abiotic stress response

Abscisic acid (ABA) is an ancient signaling molecule that is found in plants, fungi, and metazoans ranging from sponges to humans.  In plants, ABA is an essential hormone and also the central regulator protecting plants against abiotic stresses such as drought, cold, and high salinity.  These stresses—most prominently scarcity of fresh water—are major limiting factors in crop production and therefore major contributors to malnutrition.  

Malnutrition affects an estimated one billion people and contributes to more than 50% of human disease worldwide, including cancer and infectious diseases.  We have determined the structure of ABA receptors in the free state and bound to ABA.  Using computational receptor docking experiments, we have identified and verified synthetic small-molecule receptor activators as new chemical scaffolds toward the development of new, environmentally friendly, and affordable compounds that will protect plants against abiotic stresses.  We have also identified the structural mechanism of the core ABA signaling pathway through class 2C protein phosphatases and subclass 2 of Snf1-related protein kinases, which will allow modulation of this pathway through genetic engineering of crop plants.

Structural mechanism of folate and antifolate recognition by folate receptors

Folates (folic acid and derivatives) are one-carbon donors that are required for the synthesis of DNA.  Rapidly dividing cells, such as cancer cells, require high-level DNA synthesis and are therefore selectively dependent on high folate levels.  This vulnerability of cancer cells has been therapeutically exploited since the 1940s, when toxic folate analogs (antifolates) were used as the first chemotherapeutic agents.  However, current chemotherapeutic antifolates have severe side effects because they also kill non-malignant proliferative cells, such as stem cells of the bone marrow, intestinal tract, and hair follicles, resulting in immunosuppression, nausea, and hair loss. 

Cells can take up folates by two main systems, a ubiquitous, high capacity and low affinity uptake system (RFC) and by folate receptors, cysteine-rich cell surface glycoproteins which allow very high affinity uptake of folates, but not currently clinically used antifolates, by endocytosis.  While folate receptors are expressed at very low levels in most tissues, they are “hijacked” and expressed at high levels by numerous cancers.  This selective expression has been therapeutically and diagnostically exploited by administration of anti-FRα antibodies, folate-based imaging agents, and folate-conjugated drugs and toxins.  Targeting antifolates for uptake by FRs, but not by RFC, would therefore be expected to greatly reduce the side effects of antifolate chemotherapy. 

We purified human FRα lacking its C-terminal GPI membrane anchor as a mammalian-expressed, soluble IgG Fc fusion protein in the presence of folic acid and could crystallize FRα after chemically and enzymatically reducing glycosylation heterogeneity.  The crystal structure revealed a deep folate binding pocket comprised of residues that are conserved in all FR subtypes.  While the folate pteroate moiety is buried inside of the receptor, its glutamate moiety sticks out of the pocket entrance, allowing it to be conjugated to drugs without affecting FRα binding.  The structure, validated by systematic mutations of pocket residues and quantitative folic acid binding assays, provided a detailed map of the extensive interaction network between folic acid and FRα and a structural framework for the design of novel antifolates that are selectively taken up by FRs.  Our near–future goal is to determine the structures of novel preclinical chemotherapeutic antifolates bound to FRs and bound to the folate-metabolizing enzymes they inhibit to rationally design novel antifolates that are selectively targeted to cancer cells.