Rab5

Researchers discover a unique two-component molecular motor that uses a kind of renewable chemical energy to pull vesicles toward membrane-bound organelles. Cells have a fascinating feature to neatly organize their interior by using tiny protein machines called molecular motors that generate directed movements. Most of them use a common type of fuel, a kind of chemical energy, called ATP to operate. Now researchers from the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), the Cluster of Excellence Physics of Life (PoL) and the Biotechnology Center (BIOTEC) of the TU Dresden in Dresden, Germany, and the National Centre for Biological Sciences (NCBS) in Bangalore, India, discovered a novel molecular system that uses an alternative chemical energy and employs a novel mechanism to perform mechanical work. By repeatedly contracting and expanding, this molecular motor functions similarly to a classical Stirling engine and helps to distribute cargo to membrane-bound organelles. It is the first motor using two components, two differently sized proteins, Rab5 and EEA1, and is driven by GTP instead of ATP. The results are published in the journal Nature Physics. Motor proteins are remarkable molecular machines within a cell that convert chemical energy, stored in a molecule called ATP, into mechanical work. The most prominent example is myosin which helps our muscles to move. In contrast, GTPases which are small proteins have not been viewed as molecular force generators. One example is a molecular motor composed of two proteins, EEA1 and Rab5. In 2016, an interdisciplinary team of cell biologists and biophysicists in the groups of MPI-CBG directors Marino Zerial and Stephan Grill and their colleagues, including PoL and BIOTEC research group leader Marcus Jahnel, discovered that the small GTPase protein Rab5 could trigger a contraction in EEA1. These string-shaped tether proteins can recognize the Rab5 protein present in a vesicle membrane and bind to it. The binding of the much smaller Rab5 sends a message along the elongated structure of EEA1, thereby increasing its flexibility, similar to how cooking softens spaghetti. Such flexibility change produces a force that pulls the vesicle towards the target membrane, where docking and fusion occur. However, the team also hypothesized that EEA1 could switch between a flexible and a rigid state, similar to a mechanical motor motion, simply by interacting with Rab5 alone. This is where the current research sets in, taking shape via the doctoral work of the two first authors of the study. Joan Antoni Soler from Marino Zerial's research group at MPI-CBG and Anupam Singh from the group of Shashi Thutupalli, a biophysicist at the Simons Centre for the Study of Living Machines at the NCBS in Bangalore, set out to experimentally observe this motor in action. With an experimental design to investigate the dynamics of the EEA1 protein in mind, Anupam Singh spent three months at the MPI-CBG in 2019. "When I met Joan, I explained to him the idea of measuring the protein dynamics of EEA1. But these experiments required specific modifications to the protein that allowed measurement of its flexibility based on its structural changes," says Anupam. Joan Antoni Soler's expertise in protein biochemistry was a perfect fit for this challenging task. "I was delighted to learn that the approach to characterize the EEA1 protein could answer whether EEA1 and Rab5 form a two-component motor, as previously suspected. I realized that the difficulties in obtaining the correct molecules could be solved by modifying the EEA1 protein to allow fluorophores to attach to specific protein regions. This modification would make it easier to characterize the protein structure and the changes that can occur when it interacts with Rab5," explains Joan Antoni. Armed with the suitable protein molecules and the valuable support of co-author Janelle Lauer, a senior postdoctoral researcher in Marino Zerial's research group, Joan and Anupam were able characterize the dynamics of EEA1 thoroughly using the advanced laser scanning microscopes provided by the light microscopy facilities at the MPI-CBG and the NCBS. Strikingly, they discovered that the EEA1 protein could undergo multiple flexibility transition cycles, from rigid to flexible and back again, driven solely by the chemical energy released by its interaction with the GTPase Rab5. These experiments showed that EEA1 and Rab5 form a GTP-driven two-component motor. To interpret the results, Marcus Jahnel, one of the corresponding authors and research group leader at PoL and BIOTEC, developed a new physical model to describe the coupling between chemical and mechanical steps in the motor cycle. Together with Stephan Grill and Shashi Thutupalli, the biophysicists were also able to calculate the thermodynamic efficiency of the new motor system, which is comparable to that of conventional ATP-driven motor proteins. "Our results show that the proteins EEA1 and Rab5 work together as a two-component molecular motor system that can transfer chemical energy into mechanical work. As a result, they can play active mechanical roles in membrane trafficking. It is possible that the force-generating molecular motor mechanism may be conserved across other molecules and used by several other cellular compartments," Marino Zerial summarizes the study. Marcus Jahnel adds: "I am delighted that we could finally test the idea of an EEA1-Rab5 motor. It's great to see it confirmed by these new experiments. Most molecular motors use a common type of cellular fuel called ATP. Small GTPases consume another type of fuel, GTP, and have been thought of mainly as signaling molecules. That they can also drive a molecular system to generate forces and move things around puts these abundant molecules in an interesting new light." Stephan Grill is equally excited: "It's a new class of molecular motors! This one doesn't move around like the kinesin motor that transports cargo along microtubules but performs work while staying in place. It's a bit like the tentacles of an octopus." "The model we used is inspired by that of the classical Stirling engine cycle. While the traditional Stirling engine generates mechanical work by expanding and compressing gas, the two-component motor described uses proteins as the working substrate, with protein flexibility changes resulting in force generation. As a result, this type of mechanism opens up new possibilities for the development of synthetic protein engines," adds Shashi Thutupalli. Overall, the authors hope that this new interdisciplinary study could open new research avenues in both molecular cell biology and biophysics.

Neflamapimod demonstrates that it reverses the neurodegenerative process in the basal forebrain cholinergic system in animal models Phase 2a data in Dementia with Lewy Bodies show positive effects of neflamapimod on clinical endpoints associated with cholinergic neuronal function EIP Pharma plans initiation of confirmatory Phase 2b clinical trial for DLB BOSTON--(BUSINESS WIRE)-- EIP Pharma Inc., a private clinical stage biopharmaceutical company focused on developing investigative therapies for CNS diseases with significant unmet patient need, today announced publication of an article in Nature Communications showing translational results from preclinical animal models to an exploratory, placebo-controlled clinical trial of neflamapimod in patients with mild to moderate Dementia with Lewy Bodies (DLB). The findings in “Preclinical and Randomized Clinical Evaluation of the p38α Kinase Inhibitor Neflamapimod for Basal Forebrain Cholinergic Degeneration” demonstrate that neflamapimod’s unique mechanism of action has the potential to improve cognitive and motor function in patients with neurodegenerative diseases affecting the basal forebrain cholinergic system, such as DLB. Neflamapimod, which has been granted Fast Track status as a treatment for DLB by the U.S. Food and Drug Administration (FDA), is an oral, brain-penetrating small molecule that selectively targets and inhibits within the neuron the enzyme p38 MAP kinase alpha (p38α), which is known to regulate Rab5, a key protein implicated in the underlying disease process of neural degeneration in the basal forebrain. The animal studies in the publication show that neflamapimod reduces Rab5 activity and reverses the neurodegenerative process in the basal forebrain. The pre-clinical data were translated into the clinic in an exploratory Phase 2a clinical trial in DLB patients in which neflamapimod was shown to be generally safe and well tolerated, and improved versus placebo the dementia, as assessed by the gold standard dementia rating scale, and motor function. These combined pre-clinical and clinical results provide a foundation for a confirmatory, hypothesis-testing Phase 2b clinical study evaluating neflamapimod for DLB. Ralph A Nixon, MD, PhD, Director of the Center for Dementia Research at the Nathan S. Kline Institute for Psychiatric Research in Orangeburg NY and senior author on the publication, commented, “We are pleased to report on the finding of a major translational step forward towards a therapy that treats cholinergic degeneration in the basal forebrain. We hypothesized that neflamapimod would achieve this therapeutic objective because it inhibits p38α kinase, an enzyme known to regulate activity of a central player in the pathogenic process underlying cholinergic degeneration, the endosome-associated protein Rab5. Indeed, in an animal model, neflamapimod reduced Rab5 activity and reversed the neurodegenerative process in the basal forebrain. Furthermore, in the placebo controlled clinical trial of neflamapimod in DLB patients, the beneficial effects on cognition and motor function are consistent with neflamapimod treating the cholinergic deficit that underlies the clinical presentation of DLB. Taken together, the findings indicate that neflamapimod appears to represent a scientific breakthrough towards treating degeneration of the basal forebrain cholinergic system, a long-standing objective of drug development for a range of neurodegenerative diseases.” The basal forebrain is the primary source of cholinergic neural stimulation in the brain, and its degeneration occurs in aging and neurodegenerative disease-related cognitive disorders, including Dementia with Lewy Bodies (DLB), the second most common form of neurodegenerative dementia. In DLB, dysfunction in the basal forebrain cholinergic neuron (BFCN) is an important driver of disease expression and progression. Recent studies have also demonstrated that BFCN loss underpins gait dysfunction in Parkinson’s Disease. Correcting the functional deficit in the cholinergic system amplifies the release of acetylcholine and has the potential to alter the course of the neurodegeneration that drives a number of devastating brain diseases. “With this unique mechanism that treats cholinergic neuronal degeneration, in vivo preclinically and clinically in DLB patients, we strongly believe in neflamapimod’s potential to play an important role in the treatment of neurodegenerative diseases, especially in DLB where cholinergic dysfunction contributes to both the cognitive deficits and gait dysfunction that together make this a particularly debilitating disease,” said John Alam, MD, scientific founder, and Chief Executive Officer of EIP Pharma. “The cholinergic system has long been known to be critical in the pathogenesis of dementia, and neflamapimod’s pro well suited to become a foundational treatment in a disease like DLB, where there are no approved therapies. The data published today provide us confidence to progress neflamapimod into a confirmatory Phase 2b clinical trial in Dementia with Lewy Bodies.” About Neflamapimod Neflamapimod is an investigational oral, brain-penetrating small molecule drug that inhibits the intra-cellular enzyme p38MAP kinase alpha (p38α which is expressed in neurons under conditions of stress and disease. P38α plays a major role in inflammation-induced synaptic toxicity, leading to impaired synaptic function. In pre-clinical studies, neflamapimod reverses synaptic dysfunction, including within the part of the brain most affected by Dementia with Lewy bodies (DLB) – the basal forebrain cholinergic system. Results from the AscenD-LB Phase 2a clinical study demonstrated neflamapimod significantly improved cognition and function as measured by the gold standard dementia rating test, the Clinical Dementia Rating Sum-of-Boxes (CDR-SB), and also showed significant impact on motor function as measured by the Timed Up and Go test (TUG). In addition, at 40mg TID, the higher of two doses in the study, neflamapimod improved outcomes on a cognitive test battery (“Neuropsychological Test Battery”, NTB) designed to evaluate the cognitive domains most prominently impacted in DLB, attention and executive function. Neflamapimod is the first treatment that shows potential impact on cognition, function and motor function in patients with DLB. The combined pre-clinical and clinical data are consistent with neflamapimod treating the underlying disease process and having the potential to be the first disease-modifying treatment for DLB. About EIP Pharma EIP Pharma, Inc. is a private, clinical-stage biotechnology company advancing CNS-focused therapeutics to benefit patients with a range of debilitating neurodegenerative diseases with significant unmet patient need. The Company is focused on the potential of neflamapimod, an investigational compound being evaluated for its potential to treat synaptic dysfunction, the reversible aspect of the underlying neurodegenerative processes that cause disease in Dementia with Lewy Bodies and certain other major neurological disorders. For more information, please visit . Related links: Jiang, Y., Alam, J.J., Gomperts S.N. et al., “Preclinical and Randomized Clinical Evaluation of the p38α Kinase Inhibitor Neflamapimod for Basal Forebrain Cholinergic Degeneration,” Nature Communications, 13, Article number: 5308 (2022). View source version on businesswire.com: Contacts Media: Nancie Steinberg nsteinberg@lavoiehealthscience.com 917-439-2054 Source: EIP Pharma Inc. View this news release online at: