From the time that he was young, Dr. Sahil Gulati was always curious about how things worked and would perform his own experiments. That zeal for knowledge has led him to team with internationally renowned scientist Dr. Krzysztof Palczewski to make several key discoveries that could help alleviate the damage caused by several retinal and neurological degenerative diseases.
“I started mixing chemicals when I was nine years old, including mixing combustible chemicals together to make colored fire,” Gulati recalled. “I was fascinated to see how solutions with different chemical properties would mix together. For instance, watching the repelling forces a drop of soap exerts on water. That started me wondering how things really work.”
He earned a Bachelor of Technology undergraduate degree in biotechnology from Jaypee Institute of Information Technology in India. He continued his schooling there to receive a Master of Technology postgraduate degree in similar subjects. “I began with an interest in pharmacology,” stated Gulati, who was born and raised in New Delhi. “That eventually morphed into biochemistry and finally structural biology.”
Determined to earn his Ph.D., Gulati interviewed at Cornell University, University of Iowa, and Dartmouth University. He also applied to Case Western Reserve University in Cleveland, Ohio for the opportunity to work with Dr. Palczewski. “The reason I applied there was to work with him,” he recollected. “I just knew I had to work with him because of my interest in vision and neurobiology.”
Gulati joined the Department of Pharmacology at Case Western Reserve University hoping to work with Dr. Palczewski. “In the graduate program, you undergo laboratory rotations that provides a chance to work with faculty members of your choice for two months at a time. Of course, I wanted to choose Palczewski, but he was not taking graduate students at the time,” he recalled. “Instead of just accepting me into his lab, Dr. Palczewski gave me a challenge. He wanted to see if I could crystallize a membrane protein that his lab had been working on for a couple of years. If I could do it, he would take me into his lab.”
“I took the challenge. I figured ‘What is the worst that could happen?’” While trying to crystalize the protein, Gulati was also working with Dr. Phoebe L. Stewart, professor and director of the Cleveland Center for Membrane and Structural Biology. “Dr. Stewart is a pioneer in cryo-electron microscopy (cryo-EM) and an exceptional mentor” Gulati recalled. “Fortunately, I guess the stars aligned because I was able to crystallize the protein and Dr. Palczewski agreed to supervise my Ph.D. dissertation”. Finally, Gulati joined both laboratories “It turned out to be a great decision to work with Dr. Palczewski and Dr. Stewart as co-supervisors to learn x-ray crystallography and cryo-EM together and challenge myself even further.”
Antibodies, Nanobodies and Diseases
The simplest and most abundant antibody, Immunoglobulin G (IgG), constitutes about 75 percent of total immunoglobin in humans. These antibodies are involved in the innate immune system. “The recent measles outbreak in the U.S. is a result of people not being educated about vaccinations,” Gulati remarked. “Vaccines inject a lesser quantity of the virus for the body to have an immune response to. We essentially train our body to recognize the virus so when an infection happens in a larger quantity in the future, we already have the armory against that infectious agent.”
On a similar note, the use of antibodies as therapeutics is seen the light in recent years generating a market value of over $108 billion in 2017, expected to reach $218.97 billion by the end of 2023. “While delivering interesting results in the treatment of several major diseases including autoimmune, cardiovascular and infectious diseases, cancer, and inflammation, their high treatment costs impose a substantial financial burden on patients and society,” Gulati explained. “The hefty price tag comes due to research and production costs.”
What makes the work of Dr. Gulati and Palczewski so promising is that they are working with nanobodies instead of antibodies. A nanobody is a simpler protein molecule that has exactly the same effect as an IgG antibody, but is much easier to produce and is cost effective. “Nanobodies are antibody fragments which can be derived from only two species, camelid (camels and llamas) and sharks. Nanobodies are extremely effective, cheap to produce and easy to deliver as a therapy. They are on their way to being a viable class of therapeutics against several hard-to-treat diseases,” Gulati stated.
Gulati led a study that was published in Nature Communications that talks about a nanobody derived from a llama that targets signaling of G protein-coupled receptors (GPCRs), a large family of receptors involved with transmitting signals in cells. Estimates range as high as 35 percent of Federal Drug Administration-approved medications targeting GPCRs, including medications for asthma, pain, osteoporosis, and high blood pressure. “GPCRs are involved in almost everything the human body does from vision to taste to smell,” Gulati said. “GPCRs comprise only four percent of the 20,000 different proteins the human body makes. It shows how important GPCR signaling is that such a high percentage of drugs target them.”
The issue, according to Gulati, is that most small molecule and antibody-based treatments are made to target specific GPCRs. However, there are almost 1,000 different GPCRs in humans, and thereby 1,000 separate drug development pipelines will be required to target each one of them. This is an extremely expensive scenario and it will take decades of research and development to find therapies to target each GPCR.”
In the study, they targeted G proteins and not GPCRs themselves. G proteins are the immediate downstream players in GPCR signaling. Targeting G proteins can provide control on several GPCRs and might also avoid undesired cellular events. “In other words, this approach might potentially be a silver bullet for treating several medical conditions with GPCRs as key targets,” he explained. “The nanobody specifically targets a component of G protein known as Gβγ – the part that binds and efficiently activates several other signaling proteins. These proteins once activated have been linked to several types of cancers, neurological disorders and drug addiction. The nanobody binds Gβγ tightly and prevents Gβγ from activating these signaling proteins. While blocking the Gβγ signaling, the nanobody has no effect on essential GPCR signaling events that are required for normal cellular function.”
“The study serves as the first example where a nanobody has been shown to alter GPCR signaling at the G protein level by inhibiting Gβγ signaling,” Gulati said. “This will enhance the potential of nanobodies to treat various neurological conditions and cancer progression. Use of nanobodies will likely grow as research shows they are an important tool for modulating cellular signals.”
Visual Phototransduction and Retinal Diseases
The team also focuses on discovering small molecule drugs to treat retinal degenerative diseases. Recently, Gulati and Palczewski used a synthetic form of vitamin A, called locked retinal, to induce this recycling mechanism and engage proteins central to human vision. The discovery opens a new therapeutic pathway for modified retinals that help improve vision.
The discovery digs into the biochemistry of vision. Humans perceive vision with the help of an extremely sensitive protein in the back of the eye called rhodopsin, which attaches to modified form of vitamin A (retinal) to sense light. Light photons enter the eye and get absorbed by the retinal-rhodopsin complex, activating a cascade of downstream signals that constitutes our vision. Importantly, the retinal awaits light photons while maintaining a particular chemical configuration—11-cis retinal—and transforms into a second configuration—all-trans retinal—after absorption of a light photon. But this transformation is a one-way ticket and requires an army of specialized proteins to convert all-trans retinal back to 11-cis retinal. Inherited mutations in any of these specialized proteins cause retinal degenerative diseases. Researchers who want to treat vision disorders must repair or bypass the mutated proteins to maintain this retinal conversion in humans.
In the study reported in Proceedings of the National Academy of Sciences (PNAS), the locked retinal can convert one-way activation mechanism of a GPCR, rhodopsin into a controlled self-renewable cycle. “This discovery might help in alleviating the damage caused by several retinal degenerative diseases known to humans, including retinitis pigmentosa and Stargardt disease,” Gulati further explained. “Most importantly, this photo-cyclic behavior of rhodopsin bound with synthetic chromophores opens up several avenues for using optogenetic tools where light signals will be used to manipulate signaling in neurons.”
These findings exemplify the possibility of reprogramming GPCRs into self-renewing machines that can be controlled by external cues,” Gulati said. “This biochemically induced function will be helpful in treating people with vision impairment, and opens up several avenues for more efficient GPCR-based therapeutics.”
Glancing Into Off-Target Protein That Mistakenly Harbors Erectile Dysfunction Drugs
The ability for specific targeting is crucial in another study Gulati conducted, the issue of erectile dysfunction drugs causing unwanted visual side effects. Phosphodiesterase 5 (PDE5) inhibitors such as sildenafil (Viagra) and vardenafil (Levitra) are multi-billion-dollar drugs that are widely used for the treatment of erectile dysfunction and pulmonary hypertension. But, PDE5 inhibitors have also been associated with several visual side effects and, in extreme cases, damage to the optic nerve. These side effects are caused by the binding of PDE5 inhibitors to Phosphodiesterase 6 (PDE6) in the retina.
In the recent study published in Science Advances, Gulati and his group of scientists have determined the structure of the full-length PDE6 using single-particle cryo–electron microscopy. “The study provides evidence on the reorganization of fish-hook-like regions (known as the GAF β1-β2 loop regions) during the activation of PDE6 and related PDE family members, including PDE5,” he explained. The role of these fish-hook-like regions in controlling PDE activity makes them an interesting target site where a new class of PDE5 inhibitors can bind and specifically inhibit PDE5 activity with mitigated or no vision side effects. The discovery of these signal relaying hooks is promising for rational design of drug molecules that are selective to specific PDE family members and that can alleviate the side effects caused by cross-reactivity of PDE inhibitors.”
Sahil Gulati is a Life Science Application Scientist at Gatan Inc. and a former research scientist at University of California, Irvine and Case Western Reserve University.