Drug Development

Amyloid precursor protein (APP) is cleaved by the enzymes BACE and γ-secretase, which leads to the production of Aβ, which aggregates and damages neurons and is thus an important target in AD. Anti-Aβ therapeutics target increased clearance of Aβ from the brain or decrease the production of Aβ. One area of focus in the DDL is decreasing Aβ production by selectively inhibiting the APP cleavage by the enzyme BACE.

• Our goal is to find compounds that only affect the processing of APP by BACE without affecting other molecules processed by BACE.
• We screen compound libraries to identify compounds that decrease BACE activity.
• We improve compounds by making chemical variations (analogs).

As one of the key pathological characteristics of AD is the presence of amyloid plaques comprising amyloid-β (Aβ) peptide in brain tissue, we and many others would like to reduce the production and/or increase the clearance of Aβ. While Aβ plaque clearance alone has not yet resulted in significant clinical results, reduction of Aβ production itself may play a role in effective combination therapy. Aβ originates from a much larger protein named amyloid precursor protein (APP) — an apt name — and is generated by that large protein being cut (cleaved) first by an enzyme called BACE1, generating βCTF, as shown in purple above. Then a second enzyme, gamma (γ) secretase, cuts βCTF, producing Aβ that can link together to form oligomers or form large clusters that, with other components, become plaque. Because drugs that inhibit γ secretase were found to be too toxic by other groups, we targeted identification of BACE inhibitors. Our BACE inhibitors are selected on their ability to reduce BACE cleavage of APP selectively, rather than all proteins than can be cleaved by that enzyme. Inhibition of cleavage of non-APP proteins may lead to unwanted side effects, and so we are attempting to avoid this phenomenon.

In addition to being cleaved by BACE and γ-secretase to produce Aβ, APP can be processed by an enzyme called ADAM10, which leads to the production of a protein called sAPPα. Most APP is in fact processed by ADAM10. sAPPα is neurotrophic, reduces the production of Aβ, and is needed for the normal functioning of the synapses between neurons.

• Our goal is to find compounds that increase the amount of sAPPα produced in the brain.
• We have already identified a drug candidate that increases sAPPα: DDL110 (an NCE).
• We hope to find many more sAPPα enhancers by screening the 200,000-compound small molecule library at UCLA.

Perhaps our most innovative way of reducing Aβ is not by going after reduction of the activity of the enzymes that make Aβ, but by increasing a protein that is good for the functioning of synapses (sAPPα) and that, when it is increased, leads to a reduction in Aβ and could suppress the progression of the disease.

In normal, healthy brain, most APP is not cleaved by BACE and then γ secretase (although some of this cleavage is normal), but by an enzyme called "ADAM10" (shown in green). ADAM10 produces a protein termed soluble APPα (sAPPα) that is important for cell-to-cell connections in the brain. The critical cell type for this communication is the neuron, and neurons communicate through synapses. Synaptic health is supported by sAPPα.

We have already found a drug candidate sAPPα enhancer and are screening the enormous UCLA "compound library" (more than 200,000 known small molecule chemicals!) for more sAPPα enhancers. Increasing sAPPα has the additional benefit of lowering Aβ, so these compounds will have at least two effects.

Sirtuin 1 is a major longevity determinant that is found in the brain and body, and affects healthy aging.

• SirT1 is found to be lower in the blood serum of MCI and AD patients.

Sirtuin 1 is a major longevity determinant that is found in the brain and body, and affects healthy aging.

• SirT1 is found to be lower in the blood serum of MCI and AD patients.
• SirT1 levels are significantly decreased by the major risk factor for AD: ApoE4.
• We want to find compounds that normalize SirT1 levels, especially in more vulnerable individuals with the E4 form of the apolipoprotein gene.

Another well-known protein found in the brain is implicated in AD. It is an enzyme called Sirtuin 1 (SirT1), and it has been found to be lower in the brain and blood of patients with AD. This is important to know because SirT1 is a major determinant of longevity. Simply put, increasing SirT1 supports healthy aging. Furthermore, increasing SirT1 in the brain has been shown to suppress the tau pathology in an AD model. Therefore, we have targeted enhancing SirT1 in AD. An important aspect of this research is the finding that individuals who have a version of apolipoprotein designated "E4" are at greater risk for AD than people with E3 or E2 and people with E4 may have lower SirT1 even before they have symptoms of AD. We specifically target increasing SirT1 in the presence of ApoE4.

Chronic stress is associated with a higher risk for AD by increasing a chemical modification (phosphorylation) of a protein called tau, which results in ptau.

• Chronic stress is associated with a higher risk for AD.
• It increases ptau and neurofibrillary tangles, hallmarks of AD.
• We have a therapeutic that targets stress pathways and improves cognition in an AD mouse model
• We are improving this compound and will continue pre-clinical testing

Chronic stress is associated with a higher risk of AD, and there are several known physiological pathways activated or suppressed in individuals under chronic stress (but not under short-term stress). Stress increases on of the other pathological hallmarks of AD brain, neurofibrillary tangles. We already have a potential therapeutic in hand that decreases activation of one of the stress pathways involving corticotropin-releasing factor (CRF) and improves memory performance in a mouse model of AD. We are currently improving this compound and testing new compounds to identify the best one to go into pre-clinical testing.

An enzyme called neutral Sphingomeylinase2 (nSMase2) is involved in the non-canonical release of exosomes and exosome-mediated tau propagation. Brain permeable inhibitors of this enzyme could suppress the tau pathology in AD models.

• Through screening we have identified potent nSMase2 inhibitors.
• We are evaluating these inhibitors in AD models for suppression of tau propagation.

The tau pathology is implicated in the progression of AD. The enzyme nSMase2 is thought to be a key player in the spread of the tau pathology through exosome-mediated mechanisms. Brain permeable nSMase2 inhibitors would enable us to evaluate this target as a potential new therapeutic approach for Alzheimer's Disease.

In addition to BACE/γ secretase and ADAM10 cleavage, APP may be cleaved by an enzyme called a caspase to form a toxic molecule called APP-C31.

• C31 formation is increased during early AD and is associated with inflammation.
• We have identified several small molecules that inhibit the formation of C31, which we will be testing in animal models of AD.

In additional to the production of sAPPα and Aβ described on this website, under certain conditions, APP may be cleaved by an enzyme called a caspase. This produces a small fragment, APP-C31, which is particularly toxic and associated with the early pathological stages in AD. Interestingly, this cleavage seems to be upregulated at the same time inflammation increases in response to plaques, so there may be an association. We have already screened for and identified several APP-C31 lowering compounds and are ready to test them in a mouse model of AD.

It is difficult for most molecules to enter the brain because of the blood brain barrier (BBB). The BBB is created by cells surrounding the blood vessels in the brain and proteins specialized to keep toxic substances out of the vulnerable brain.

• We use pharmacokinetic studies to determine if a compound gets into the brain.
• For molecules that are not brain-penetrant, we have created an SE delivery system using microfluidic synthesis which consists of small deformable nanovesicles (DNV) that can encapsulate drugs and potentially be used to deliver small molecule drugs, protein therapeutics, antibody therapeutics, mRNA and other molecules across the BBB and into the brain.

AD is a disease of the brain, but it can be difficult for many otherwise promising therapeutics to get into the brain due to the blood-brain-barrier (BBB). It is formed by the cells that line the blood vessels and capillaries in the brain, and only certain compounds can get in. To assist with entry of molecules into the brain that would otherwise be excluded, we are using our microfluidic reaction technology to create small membranous particles — liposomes — in which a compound can be encapsulated for brain delivery.

Exosomes are membranous particles that are secreted by cells and contain proteins cellular components that reflect the physiological state of the cell.

• They can be found in body fluids, such as blood, saliva, and cerebrospinal fluid.
• We have the ability to isolate exosomes that come specifically from the brain.
• We are actively pursuing the use of neuron-derived exosomes to determine the level of alertness and to gain information regarding disease state in neurodegenerative disorders.
• We also hope to develop methods to use exosomes to follow responses to drug treatment.

We make our nanovesicles to enhance drug delivery to the brain artificially, but the cells of the body and brain make their own particles that are structurally very similar. These vesicles, called exosomes, can contain cellular components that reflect the physiological state of the cell. Exosomes are expelled from cells and can be found in cerebrospinal fluid, blood, urine, and even saliva. Analysis of these exosomes could be very useful if we can determine the cell type and organ (such as the brain) from which they came. We therefore are developing methods to detect this information. Specifically, we have work underway to use exosomes in saliva to determine state of alertness (cognitive fatigue), and a separate study to use exosomes in plasma to predict disease recurrence.

This new cutting-edge method can be used to test the effects of therapeutics in differentiated cells derived from pluripotent stem cells originating from patients with different disease characteristics and genetics.

• Skin cells from patients can be turned into stem cells and then into neurons.
• These stem-cell-derived neurons can then be treated with different therapeutic compounds to test whether cells from different patients respond differently to the same therapeutic.
• Different compounds can be compared for neurons from the same donor.
• We can then tailor clinical trials of these compounds to include only patients whose stem-cell-derived neurons were responsive to the therapeutic. This would be a personalized medicine.

One of the most cutting edge methods the Drug Discovery Lab is beginning to utilize is a "clinical trial in a dish". This method, as described by Haston and Finkbeiner, and others, involves isolating skin cells from a variety of patients with known genetic differences, and transforming these cells into a type of stem cell — an "induced pluripotential stem cell". As the name implies, these cells can become a variety of different cells types if guided in a specific way. For AD, that might be differentiation into cortical neurons. These neurons then, derived from real and varied human patients, would be treated with a potential therapeutic to see its effects. If the effects differ in cells from individuals, and if a future clinical trial reveals the trial in a dish can be predictive of efficacy, it could then be used to select the right patients for the right drug treatment. This is a form of personalized "macro-medicine".