UCSF

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Structure, function, and modulation of protein-protein interactions and protein networks

Our lab has been interested in how proteins recognize each other and how we can use this information to develop inhibitors of protein-protein interactions. Recently, we have taken on the broader challenge of understanding how proteins interact in networks to modulate arrays of cellular activities. Inhibiting or stabilizing one interaction might have global effects on these interacting networks. Our long-term aim is to develop context-specific modulators of protein-protein interactions to interrogate biology and develop innovative new medicines.

Allosteric and protein-protein interaction modulators of p97

The AAA+ ATPase p97 is a master regulator of proteostasis and is involved in functions as diverse as chromatin remodeling, ER-mediated degradation, and golgi remodeling. p97 forms a homohexamer with a three-domain architecture: an N-terminal domain that functions as an anchor site for cofactor/substrate recognition and two ATPase domains D1 and D2. The D1 and D2 domains form two stacked rings, creating a central pore through which unfolded protein strands can pass. p97 is reported to interact with up to forty regulatory enzymes and adaptor proteins. Each directs p97 to a specific protein quality control pathway. Due to p97’s important role in maintaining proteostasis, it is implicated in cancer, neurodegenerative disease, and the degenerative disorder multisystem proteinopathy (MSP1, also called IBMPFD). Using small molecules and protein engineering, we aim to selectively influence p97-mediated activities, protein-protein interactions, and cellular function.

Structural studies in our laboratory and several others has revealed at least three distinct conformations of p97 depending on the identity of bound nucleotide, the presence of disease-causing mutations at the ND1 interface, and perhaps protein-protein interactions. These conformations, as visualized by cryo-EM, are shown in the movie below. This movie iterates among the three major, symmetrical conformations observed in the static cryo-EM reconstructions. In these images, the D2-domain ring undergoes a contraction between the ATP- and ADP-bound states; ATPase-dependent structural changes provide a potential mechanism for threading unfolding protein through p97’s central pore. Also, there is a major conformational change in the relationship between the N-domains and D1 ATPase ring. When ATP is bound to D1, the N-domains adopt an “up” conformation above the plane of the D1 ring; when ADP is bound, the N domains lie in the plane of the ring (2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition, Science, 2016).

diagram

p97 is involved in diverse cellular activities.

We have employed biochemical and biophysical techniques to understand the interplay between nucleotide-binding, cofactor-recognition, p97 conformational change, and age-related cellular changes such as pH in the function of p97. These outcomes will provide insights into small molecule drug discovery for this protein target.

Small-molecule modulators of p97 function

Working with members of the NCI Chemical Biology Consortium (U Pittsburgh, U Minnesota, UCLA, Caltech, SRII), we have developed potent, allosteric inhibitors of p97. High-resolution structures of p97 bound to UPCDC30245 indicate that the compound binds to the ADP-bound state and blocks ADP release (2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition, Science, 2016). We have also identified molecules that bind to regulatory subunits on p97, including one series that binds to the N-domain and modulates p97/adaptor PPI (A Fragment-Based Ligand Screen Against Part of a Large Protein Machine, J. Biol. Screen, 2015). Future work will optimize these compounds and reveal their mechanisms of action in biochemical and cell-based models.

cryo EM structure

Biochemical exploration and manipulation of p97-mediated protein-protein interactions

A network of regulatory binding partners recruits p97 to specific cellular tasks. Some are adaptors that help p97 recruit ubiquitinated protein clients. Others are enzymatic cofactors (such as deubiquitinases, ligases, and proteases) that help p97 process clients. It is unclear how p97 works with its binding partners to perform its myriad cellular functions. We are investigating whether nucleotide-driven p97 conformational changes alter its interactions with cofactors and adaptors and drive its activity.

diagram

Cofactors and adaptors may prefer different p97 conformations and direct p97 to different cellular activities.

We hypothesize that conformational switching in p97 structure regulates its adaptor binding, thereby altering the PPI network and thus cellular functions of p97. For instance, we have obtained preliminary evidence for preferential binding of two adaptor proteins (p47 and p37) in the “up” conformation of p97 (p97 Disease Mutations Modulate Nucleotide-Induced Conformation to Alter Protein–Protein Interactions, Bulfer, et al, ACS Chem Biol, 2016); other labs have also considered the conformational preferences of other adaptor proteins. Our lab is further extending this study to diverse p97-adaptor proteins involved in a variety of cellular functions such as ER-associated degradation (ERAD), autophagy, and membrane fusion. We are also developing conformational locks of p97 and will use these tools to determine the effect of p97 conformational switching on adaptor binding and function in cells.

To further advance our understanding of the PPI network mediated by p97, we are also interested in engineering adaptor-specific p97 using phage display. This will allow us to study the downstream cellular effects of such a blockade and understand the cellular role of the individual p97-adaptor complex in greater detail.

Completion of these aims will help us resolve how recruitment of different adaptor proteins to p97 is regulated in vitro as well as in vivo, and to understand the role of ATP hydrolysis in p97’s various cellular functions.

Allostery and protein-protein interactions of integrin I-domains

Integrins are large heterodimeric adhesion receptors expressed on the surface of all eukaryotic cells and interface the cell with its environment. The αM (Mac1) integrin, found on the surface of innate immune cells such as macrophage, microglia, and neutrophils, binds to ligands using an Inserted (I) domain. Of particular interest, Mac1 binds to the clotting protein fibrin(ogen), activating innate immunity in response to vascular leakiness in multiple sclerosis (Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation, Ryu, et al, Nat Comm, 2015).

Shown in the following figure, Mac1 and its homolog αL (Lfa1) undergo an allosteric shift of the c-terminal α7 helix after binding of an protein partner to their Metal Ion Dependent Adhesion Site (MIDAS). In Lfa1, the downward register of the α7 helix can be blocked with small molecules which bind underneath the α7 helix and result in decreased affinity for protein partners.

alpha-7 helix in Mac1

The alpha-7 helix in Mac1 shifts between inactive (red) and active (blue) states.

To understand the allosteric coupling between the MIDAS and the α7 helix, as well as to pursue allosteric inhibitors of this and other αI-domains, we compared the ligandability of the Lfa1 and Mac1 αI-domains using high-throughput disulfide tethering (see below). We find that the α7 helix pocket is more ligandable in Lfa1 than in Mac1, likely due to differing dynamics in the protein structure. However, we were able to identify one compound that binds in the allosteric pocket, using a technology called tethering (see below).

Tethering and targeting cysteine reactivity

Disulfide trapping is a fragment screening method that allows the targeting of specific sites of interest on proteins. In this approach, a protein containing a native or introduced cysteine residue near a site of interest is screened against a library of thiol-containing, drug-like fragments under conditions that promote thiolate-disulfide exchange. Fragments that form both noncovalent and covalent binding interactions near a cysteine residue are stabilized and therefore persist, whereas fragments that bind non-specifically are reduced away.

The fragment-modified protein is stable and is readily detected by high-throughput mass spectrometry, allowing identification of the bound fragment. By screening pools of distinct-mass fragments, thousands of fragments can be screened per week. The low hit rate observed (about one to five percent) speaks to the selectivity of the method and permits the screening of proteins containing multiple thiols without multiple fragment binding. Crystallographic studies have demonstrated that disulfide-trapped fragments bind in the same mode as the corresponding free (untethered) fragment, thus validating this approach for the design of noncovalently bound ligands.

diagram

In the figure above, multiple potential pathways exist to optimize candidate fragments for potency, selectivity, and cell-based activity. The noncovalent portion of the fragment can be optimized through structure-guided design and/or empirical SAR. Using an “extended tethering” approach, the first fragment is modified to include a new thiol that can serve as new “bait” to screen for a second fragment that binds in a nearby site; converting the resulting disulfide bond leads to a high-affinity ligand (top). If the coordinating cysteine residue is present in the wild-type protein, the disulfide can also be converted to a weak electrophile (center); we are exploring this approach for non-catalytic cysteine residues near substrate-binding sites. Finally, if a second fragment/ligand is known to bind nearby, the two fragments can be linked to form a high-affinity, noncovalent compound for further medicinal chemistry (bottom).

See also:

Novel inhibitors of caspase-6

Caspases play key roles in inflammation, apoptosis, and neurodegeneration. However, it has been very difficult to identify cell-active, selective caspase inhibitors. Caspases-2 and -6 are particularly implicated in neuronal development, axonal pruning, and excitotoxicity. Caspase-6 is also associated with cleavage of tau protein in Alzheimer’s disease (AD); tau cleaved by Caspase-6 has been found in neurofibrillary tangles and in brains with mild through severe forms of AD, and elevated levels of active Caspase-6 has been found in AD brains.

To address the role of caspase-6 in neurodegenerative disease, we set out to develop highly selective and potent cell-active probes of caspase-6. In collaboration with the Renslo Lab and Genentech, we identified potent noncovalent inhibitors of caspase-6 that exhibit exquisite selectivity over other caspases by binding uncompetitively to the enzyme/substrate complex (Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6, Heise, et al, PLoS One, 2012).

Our current work is focused on probes that bind covalently to caspase-6. Using our disulfide-trapping technology, we identified covalent inhibitors that bind to a non-conserved cysteine residue in the caspase-6 substrate-binding groove. Working with the Renslo Lab, we have optimized electrophilic analogs that show >1000-fold selectivity for binding/inhibiting caspase-6 over other caspases. We solved co-crystal structures of compounds bound to caspase-6 and are using structure to design more potent inhibitors. Current work is focused on demonstrating cellular activity and selectivity. If successful, we will use these compounds to address the fate of tau and neurons with active and inhibited caspase-6.

Probe and drug discovery using high-content imaging

High-content analysis (HCA) is a powerful assay methodology that combines high-throughput microscopy and automated image analysis. We hypothesize that cell-based screens designed with primary cells will more faithfully translate to model organisms and humans than do screens designed in cell-culture lines. Towards this aim, we have developed HCA for human hematopoietic stem cells, primary immune cells from brain, and ciliated fibroblasts. Another focus is screening whole parasitic organisms (Giardia, T. cruzi, Leishmania, S. mansoni), developed in partnership with the Center for Discovery and Innovation in Parasitic Diseases (CDIPD).

Hematopoietic stem cell expansion

Hematopoietic stem cell (HSC) transplantation is a life-saving therapy for malignant and nonmalignant blood diseases, yet only about 20% of patients can find a matched, related donor. Umbilical cord blood (UCB)-derived HSCs are ideal due to their high donor-recipient mismatch tolerance, yet the majority of banked UCB units have too few cells for clinical use in adults. To address this need, we are developing small molecules capable of expanding HSCs ex vivo for clinical transplantation.

In conjunction with the Leavitt Lab at UCSF and the SMDC HTS team, we conducted a high-throughput, high-content imaging analysis screen using primary human HSCs, and identified and optimized three chemical series that greatly expanded the number of multipotent cells. In collaboration with The Jackson Laboratory we are testing the in vivo engraftment potential of compound-expanded cells in mouse xenograft models. We are currently investigating the mechanism of action of each of these classes of HSC expanders to explore the underlying biology and to develop these compounds and our culture system into a clinically useful therapy.

diagram

Live imaging of Schistosoma

Collaborating with Conor Caffrey, PhD (UCSD), we are studying the causative agent of schistosomiasis. Schistosomes are parasitic flatworms that infect 3.5% of the world’s population yet have been recalcitrant to modern methods of drug discovery. We have developed an automated high-content screening platform to quantify chemically-induced, multi-parametric responses of living Schistosoma mansoni. We demonstrated the importance of measuring motility in defining drug phenotypes/mechanisms and provided unprecedented time- and dose-quantitation. We aim to develop drug screening for helminths as a fully quantitative science and to provide tools to explore parasite development and drug mechanism of action.

Schistosomula

Schistosomula, the youth-stage of the human parasite S. mansoni, swimming to the center of a well in a 96-well plate.

High-content imaging tools provide the opportunity to study high-resolution biology in a high-throughput and quantitative format. Using complex cell culture and whole organisms, we aim to solve biological problems and also to advance high-content imaging technology.