science

Cell Chemistry Gone Wrong: How Disease Begins in Cells and How We Can Fix It

The internal chemistry of human cells is a marvelously complex world.

Cells have DNA. DNA makes enzymes (skip a step or two). Enzymes chaperone chemical reactions in the cell. That chemistry determines cell function, cell health, cell coordination with other cells and tissues, and much else.

Luciferase is an enzyme that fixes oxygen to a substrate to produce light; the beauty of both fireflies and jellyfish depends on this. Rubisco is an enzyme that fixes carbon; plants need it to convert air into sugar. Enzymes are used in chemical pathways that cascade into the mundane (mitosis and meiosis, cellular respiration), the species-specific (vitamin C synthesis native to every mammal save humans and cavies), the history-making (bread and beer fermentation), the tech savvy (biofuel production, artemisinin synthesis), and more.

If you’re studying the current hot button research topics of human health right now, there are some pathways that come up a lot. There’s the MAPK pathway involved in human cell division; tumors form when it goes awry. The insulin signaling pathway is connected to body weight and glucose (sugar) regulation. Now, these pathways are incredibly complex – there are lots of enzymes involved. We are only now untangling many of the enzymes and their effects in these systems, and we can inhibit ones we think are causing the disease when, for some reason, there’s too much of it present.

In these pathways, a phosphate group serves as a “tag.” The phosphate group is a single phosphorus atom surrounded by four oxygen atoms, and it can be attached or detached to other molecules. We call this phosphorylation and dephosphorylation, and it is a common signaling technique cells use. A phosphorylated molecule is saying “pick me!” or “use me to reach the next step!” Then, when the phosphate group is removed, that signal goes away.

A phosphate group serves as an on signal for a molecule, activating the next step in a chemical reaction.

This is a good thing! In the insulin signaling pathway, there are times when you want it on, and times when you want it off. Insulin, you may know, is a hormone used to regulate sugar in the human body. Diabetes is a disease that can occurs if insulin becomes imbalanced. If insulin is imbalanced, that’s because its chemical pathway isn’t being regulated properly.

Kinases and phosphatases switch steps of a chemical pathway on and off, so they are likely culprits. As it turns out, molecules that inhibit activity of these enzymes can be potent drugs for treating diseases from chemical pathways gone awry. And while clinical kinase drugs exist and are being used, we still haven’t found any phosphatase-targeting drugs – yet.

Why target phosphatases? There are lots of reasons. There’s this one phosphatase enzyme named PTP1B. Its increased activity can cause tumor formation in mammary glands, resulting in breast cancer. It’s also associated with neuron loss, inflammation, and lost spatial memory in mice – all of which are symptoms of Alzheimer’s disease. Finally, PTP1B can mess with signaling pathways relating to metabolism, such as the insulin pathway. That makes it a double whammy, because it makes it a prime target for obesity and diabetes, and also a target again for Alzheimer’s, since people with Type 2 diabetes are at a much higher risk for Alzheimer’s and dementia.

Clearly, an inhibitor for PTP1B would have the potential to inform new drug design for multiple disorders. However, most of the inhibitors we’ve found for it so far target the active site of the enzyme. That’s a problem, due to some of the chemistry of PTP1B.

A competitive inhibitor binds to the active site, blocking the enzyme from binding the substrate. An allosteric inhibitor binds to an allosteric site, changing the enzyme’s shape so it can’t bind the substrate.

We call the molecules that enzymes bind to “substrates” – they are the substrate the enzyme acts on to do its thing. The active site is where the substrate binds to an enzyme. In PTP1B, the active site has a positively charged amino acid that clings the substrate. So any inhibitor that binds the active site (beating the real substrate in this chemical binding race) will have to be negatively charged. Negatively charged molecules are not good pharmaceutical candidates. That kind of chemistry makes them hard for cells to take up. Plus, there are lots and lots of phosphatases – PTP1B is only one of them. Most phosphatases have very conserved active sites. That means that their active sites are very similar, so an inhibitor that binds PTP1B will also bind the active site of good phosphatases. Considering that there are phosphatases that help regulate your immune system, a nonspecific inhibitor would wreak havoc on the human body!

The structure of PTP1B changes when it is active or inactive. If an inhibitor is present, it twists the enzyme into its inactive, “closed loop” form. Based on Fig. 3b from Wiesmann et al. 2004.

Lucky for us, enzymes are more than their active sites. Enzymes are large, complicated molecules, and when it comes to phosphatases, they really start to differ from each other in their non-active regions. An inhibitor can also bind to an allosteric site. This means it’s binding somewhere else on the enzyme. Instead of physically blocking the substrate from reaching the active site, it changes the active site so that binding is no longer possible! We have actually seen this happen with allosteric inhibitors that bind PTP1B. We know what PTP1B looks like when it’s active – it has a nice big loop for the substrate to enter. When it’s inactive, that loop closes. An allosteric inhibitor that binds PTP1B will convert it into the inactive shape. Such inhibitors don’t typically have a charge and are much better candidates for future pharmaceuticals.

So, why not scope out the allosteric inhibitor space? That’d be a great strategy for finding these amazing new drugs, right? Well, we still don’t know all that much about phosphatase structure, or where the allosteric sites are. That missing information makes finding molecules that will bind those allosteric regions very difficult. To date, there are no phosphatase inhibiting drugs that use this strategy, or even drugs for treating phosphatase activity at all.

Is there a way to find small molecule inhibitors without knowing where and how they should bind? Well, natural products are a good source here. Nature is very good at designing small molecules with very specific chemical properties, and we’ve used them for drug discovery before. But nature designs those molecules based off of survival value. It just so happens that often their survival value offers us an unexpected health benefit. How do we get a living system to evolve a chemical property that is only something we want? Can we set up living systems so that they will evolve allosteric phosphatase inhibitors so we could then pick and choose from a set of new drug targets?

Well, yes, actually. We can build living systems to discover small molecules we are interested in. We may not know what those molecules should look like, but a cell doesn’t care about that. It only cares about function. How do you build a cell to link survival to any desired chemical activity, forcing it to evolve the kinds of molecules you need? That is a question for another day.

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