Parkinson’s Disease (PD) is a debilitating neurodegenerative disorder involving a loss of the critical neurotransmitter dopamine, due to death of neurons in specific brain regions, most notably the substantia nigra in the midbrain. The pathophysiological mechanisms implicated in PD are complex and diverse. Thus, as for many complex diseases, it is likely that different molecular defects account for PD in specific subsets of PD patients. Prosetta’s distinctive approach to subcellular biochemistry leads to a new perspective on PD pathophysiology and suggests novel approaches to its treatment, for which we are advancing small molecules.
The enzymes of a biochemical pathway are often together in multi-protein complexes (MPCs) that allow these enzymes to function in the coordinated manner crucial for homeostasis. A key insight of the Prosetta paradigm is that to understand a biochemical pathway you need to study not only its specific component enzymes, but also the (previously unappreciated) assembly machines that are involved in the catalyzed bringing together (assembly) of these enzymes into an MPC. Some of the most prevalent forms of many common diseases, including PD, appear to involve molecular defects in MPC assembly. Long viewed as occurring through spontaneous self-assembly, Prosetta studies suggest assembly to be catalyzed – by the assembly machines we have discovered, and for which we have drug-like small molecule modulators.
One way in which assembly can be crucial for disease pathogenesis is when disease-triggering insults (environmental, genetic, or epigenetic) cause the formation of aberrant assembly machines. The consequence of assembly machine dysfunction is a defective assembled product, and when that product is a biochemical pathway’s MPC, the pathway is dysfunctional. There may be many manifestations of such pathway dysfunction including dysregulation of the expected enzymatic activity, appearance of new enzymatic activities, altered feedback, -- or protein aggregation. Some of these may lead to chronic stress/inflammation and have myriad further effects. Each of these consequences can culminate in disease of varying time scales and magnitude.
We hypothesize that the allosteric sites that govern assembly machine composition and action are excellent targets for next-generation pharmacotherapy, for two different reasons. First, because they address the underlying primary molecular cause of diverse disease phenotypes, rather than responding simply (and in futile fashion) to phenotypic consequences. Second, because they promise a more physiological approach to pathway modulation, thereby diminishing unintended consequences ensuing from conventional pharmacologic pathway blockade (e.g. side effects). For these reasons we have focused on discovery of drugs that modulate assembly to correct aberrant assembly machines and/or their actions, via their allosteric sites, rather than on inhibition of their individual protein components. The outcome of that effort has been quite productive, and drug-like compounds with those characteristics have been identified and are being advanced for a variety of disorders including PD.
Viruses are extremely efficient manipulators of evolution since their generation time is very much faster than that of their host cells. Thus, screens for small molecules that block viral assembly provide the path by which we have identified cellular MPCs that govern cell homeostasis. Remarkably, we are also able to drive structure-activity relationship optimization to identify compounds that, likely due to achieving aberrant assembly machine specificity, modulate assembly machine composition independent of the signaling dictates of viral assembly. In the Prosetta paradigm, these cellular MPCs are of importance in non-viral diseases, including in neurodegeneration. Hence, drugs blocking the allosteric sites of aberrant MPCs identified by an antiviral screen can also serve as initial hit compounds whose advanced lead series analogs are more selective for non-viral disease cellular pathology.
Prosetta has used its cell-free protein synthesis and assembly (CFPSA) system, applied to viral assembly, to create a collection of drug-like small molecules that target assembly from each of most viral families that cause human disease. This compound collection, some 300 chemotypes distilled from a larger library of 150K compounds, has been termed the Prosetta “Hit-finder” collection (HFC). This collection can be considered a mirror of cellular MPCs involved in cellular homeostasis and pathology.
Prosetta’s approach to complex diseases including PD and other neurodegenerative disorders, is to first attempt to assemble in an energy-dependent manner de novo synthesized proteins implicated in the disease. Once conditions to assemble a large complex of relevant proteins (or subset thereof) are defined, the pathway by which that complex is formed can be probed with the small molecules of the HFC. Alternatively, a cellular model for the disease in question can be used to directly screen the HFC compounds for biologically plausible therapeutic effects. The relatively small number of assembly-modulating compounds comprising the HFC allows difficult cellular, or even animal model studies to be carried out that would be impractical to apply to larger compound collections. Once such biological plausibility of therapeutic effect in a credible cellular model has been established, synthesis and assessment of analogs is straightforward.
Following demonstration of a robust SAR, drug resin affinity chromatography (DRAC) and photo/chemical crosslinking (P/CCL) can be applied to transition from a target-agnostic phenotypic screen to identify the MPC target and nearest neighbor proteins of the drug binding site. At that point, DRAC can also be used to assess target engagement. This approach also allows Prosetta to use SAR advancement to drive to aberrant assembly machine specificity as a means of solving the paradox of how to target the host without host toxicity. Aberrant assembly machines are not homeostatic and therefore their destruction does NOT carry host liabilities, even though they are composed of host proteins. Finally, once a target product profile (TPP) is reached, an animal efficacy trial can be conducted and an investigational new drug (IND) application with the FDA filed. Prosetta is approaching TPP in several therapeutic areas including a number of anti-viral programs and ALS.
In the case of PD, working closely with its academic collaborators in the laboratory of Professor Carsten Korth, Dusseldorf, Germany, Prosetta has identified a number of HFC compounds that modulate a-synuclein (a-SYN) assembly in ways that are neuroprotective. A subset – but only a subset — of the a-SYN-active HFC compounds are also active in other neurodegenerative disorder cellular models including ALS (re-localization of TDP-43 to the nucleus; elimination of TDP-43 aggregates in stress granules) and AD (Abeta oligomerization; Tau phosphorylation). This suggests that diverse protein aggregation disorders may include subsets due to shared assembly machines and/or similar allosteric sites. Further work will be needed to determine whether the optimal compounds for advancement are those that are selective for one vs another assembly target. Efforts are currently underway not only to advance these compounds, but also to develop the peripheral biomarkers by which patients could be stratified into subsets for which particular assembly modulator compounds are most effective.