Biology subjects

Hariharan, P.

Publications and source records attributed to Hariharan, P..

7 recordsLinked to original sources

bioRxiv2025

Guiding treatment response by spatiotemporal control of α-particle deposition in solid tumors: the case for 'affinity cocktails' of antibody-radioconjugates

Antibody-radioconjugates are leading the investigational targeted alpha-particle (-particle) therapies for the treatment of solid tumors that do not respond to approved therapies. Yet, there is still treatment failure in the clinic largely attributed to the heterogenous patterns of tumor irradiation by -particles. Although -particles are essentially impervious to resistance, attributed to the complex double-strand DNA breaks they cause while traversing cells, cells not being directly hit by -particles will likely not be killed. The diffusion-limited poor tumor penetration of high-affinity (strongly-binding) antibody-radioconjugates combined with -particles short-range in tissue (only 40-80m), let tumor regions far from vasculature inadequately irradiated, therefore, possibly escaping treatment. METHODSTo improve penetration of delivered activity within tumors, we engineered separate actinium-225 antibody-radioconjugates of variable affinities ( affinity cocktails) targeting the same marker on cancer cells, that were chosen based on their preferential irradiation of complementary regions of the same tumors. The cocktails comprise: (a) high-affinity antibody-radioconjugates (as the ones on clinical trials), which mostly deliver their cargo in tumor cells close to the vasculature, where the low(er)-affinity antibody-radioconjugates fail to deliver effective doses, due to their fast clearance; and (b) low(er)-affinity antibody-radioconjugates, that penetrate the deeper parts of tumors farther from the vasculature, where the high-affinity antibodies fail to reach. The efficacy of affinity cocktails was assessed in spheroids, that were employed as surrogates of tumor avascular regions, and on mice with subcutaneous xenografts of different cancer origin, expression levels and/or type of the targeted receptor: HER2 highly-expressing BT-474 breast cancer cells, HER2 moderately-expressing HEPG2 hepatoma cells, and/or HER1 low-expressing BxPC-3 pancreatic cancer cells. RESULTSAlthough the high-affinity antibody-radioconjugates were most lethal against cancer cells in monolayers, affinity cocktails were most effective in inhibiting spheroid growth, due to better collective spreading of the antibody-conjugates within the spheroids volume. On all mouse models, and for the same total injected activity, affinity cocktails resulted in the best tumor growth inhibition, even at lower tumor absorbed doses, compared to the high-affinity antibody-radioconjugates alone. CONCLUSIONSThis proof-of-concept study in -particle antibody-delivery to solid tumors demonstrates that separating the two key processes of diffusion and reaction/binding improves treatment efficacy. This generalizable approach may augment antibody-radioconjugates already in clinical trials.

bioengineering↗

bioRxiv2025

Actinium-225 dendrimer-radioconjugates combined with low-dose standard-of-care chemotherapy: site-independent treatment of triple negative breast cancer metastases

PURPOSEMetastatic triple negative breast cancer (mTNBC) is incurable largely due to the development of drug resistance, the lack of selective cell targeting, and/or limitations in tumor drug delivery that vary depending on the different (metastatic) tumor locations. METHODSThe potential of a single type of systemic, targeted alpha-particle therapy (TAT) was investigated for addressing the above challenges of TNBC tumors implanted at different anatomic sites in mice. Actinium-225 dendrimer-radioconjugates alone, and/or after pretreatment with low-dose standard-of-care cisplatin, were assessed in vitro and on immune-competent 4T1-Balb/c mouse models with tumors implanted intracranially, orthotopically or subcutaneously. RESULTSIn vitro, TATs efficacy was enhanced by cisplatin. In vivo, treatment was initiated well after tumors had grown (Vto = 39 {+/-} 14 mm3 in the intracranial model, and Vto=100mm3 in the orthotopic and subcutaneous models). Across all tumor implantation sites, a unified correlation was observed between animal mean survival and the dendrimer-delivered tumor absorbed doses, which were selectively increased by low-dose cisplatin pretreatment. Importantly, in all animal models, the mean survival following systemic treatment with both modalities was significantly longer vs. each modality alone and/or vs. no treatment, at injected doses that did not cause long-term (10-month) toxicities in tumor-free mice. CONCLUSIONSystemically-injected dendrimer-delivered TAT, combined with low-dose cisplatin pretreatment, can safely extend survival independent of mTNBC tumors anatomic site, potentially presenting a single type of therapy to simultaneously treat multi-site mTNBC.

bioengineering↗

bioRxiv2025

Allosteric effects of the coupling cation in melibiose transporter MelB

The major facilitator superfamily (MFS) transporters play significant roles in human health and disease. Salmonella enterica serovar Typhimurium melibiose permease (MelBSt), which catalyzes the symport of galactosides with Na+, H+, or Li+, is a prototype of this important transporter superfamily. We have published the structures of the inward- and outward-facing conformations of MelBSt with galactoside or Na+ bound, determined the binding thermodynamic cycle, and proposed that positive cooperativity between the two co-transported solutes plays a key role in the symport mechanism of MelBSt. The molecular basis for this core mechanism remains unclear. In this study, we determined the molecular basis for this core symport mechanism through analyzing the structural dynamics of MelBSt and effects induced by melibiose, Na+, or both using hydrogen-deuterium exchange mass spectrometry (HDX-MS). We also refined the specific determinants for the sugar recognition in both protein and galactoside molecules by solving the crystal structures of a uniporter D59C MelBSt bound to melibiose and other sugars, and identified a critical water molecule as part of sugar recognition. Our integrated studies from structure, HDX-MS, and molecular dynamics simulations support the conclusion that sugar-binding affinity is directly correlated with protein dynamics. The binding of the coupling cation at a remote site functions as an allosteric activator to restrain the conformational flexibility of dynamic residues in the sugar-binding site and in the cytoplasmic gating salt-bridge network, thereby increasing sugar-binding affinity allosterically. This study provides a molecular-level schematic of the fundamental symport mechanism via positive cooperativity, which may serve as a general mechanism for cation-coupled symporters.

biophysics↗

bioRxiv2024

Glioblastoma Treatment by Systemic Actinium-225 alpha-particle Dendrimer-radioconjugates is Improved by Chemotherapy

RATIONALEThe poor prognosis of glioblastoma is largely due to drug resistance and tumor location that, together, make it difficult to treat aggressively without affecting the rest of the brain. METHODOLOGYHigh-energy, short-range (40-80{micro}m) dendrimer-delivered -particles could address both challenges, because (1) they cause complex, highly cytotoxic double-strand DNA breaks, and (2) irradiation of the neighboring brain is minimal, since dendrimers selectively delivers them to tumors. Since cancer cells that are not directly hit by -particles will likely not be killed, the patterns of tumor irradiation affect efficacy. Systemically injected dendrimers extensively accumulate in glioblastomas, where they are taken up by tumor associated macrophages (TAMs), which tend to infiltrate tumors. We hypothesized that dendrimers labeled with -particle emitters, when being carried by TAMs, could more evenly irradiate glioblastomas, improving survival. In this study, the efficacy of dendrimers radiolabeled with the -particle emitter actinium-225 (dendrimer-radioconjugates) was evaluated when administered alone and/or after temozolomide, in a syngeneic immune-competent orthotopic GL261-C57BL/6 mouse model. RESULTSSystemically-administered dendrimer-radioconjugates, at activities that did not result in long-term toxicities, prolonged survival of mice with orthotopic GL261 tumors, compared to standard-of-care temozolomide (39 vs 31 days mean survival, p=0.0061) and non-treated animals (30 days, p=0.0009). Importantly, injection of temozolomide 24 hours before administration of dendrimer-radioconjugates further improved survival remarkably (44 days). This improvement in efficacy was attributed to: (1) the significant increase (by 33%) in tumor absorbed doses delivered by dendrimer-radioconjugates when injected after chemotherapy, without altering normal organ dosimetry, while sparing the tumor-surrounding healthy brain; (2) the potentially deeper tumor penetration of dendrimer-radioconjugates, suggested by the enhancement of dendrimer penetration within GL261-spheroids, employed as model tumor-avascular regions and/or TAM-free regions; and/or (3) the formation of a more lethal cocktail when both modalities acted on same cancer cells, that was correlated with increased levels of dendrimer-radioconjugates associating with GL261 cells in vitro and with greater incidences of karyomegaly in vivo. CONCLUSIONSThis study demonstrates the potential of a brain tumor targeted systemic actinium-225 radiopharmaceutical therapy that inhibits growth of glioblastoma cells and prolongs survival of mice with orthotopic brain tumors, further improved by standard-of-care temozolomide, without notable toxicities.

bioengineering↗

bioRxiv2024

Distinct roles of the major binding residues in the cation-binding pocket of MelB

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with either H+, Li+, or Na+, but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt, as well as the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport still remain poorly understood. We have solved two x-ray crystal structures of MelBSt cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published previously. We determined the energetic contributions of three major Na+-binding residues in cation selectivity for Na+ and H+ by the free energy simulations. The D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport with poor activities at higher {Delta}pH and better activities at reversal {Delta}pH was observed, supporting that the membrane potential is the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket.

biophysics↗

bioRxiv2023

Mimicking the regulatory state of a major facilitator superfamily sugar transporter

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na+-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. Collectively with the available outward-facing sugar-bound structures, both the outer and inner barriers were localized. The N-and C-terminal residues of the inner barrier contribute to the sugar selectivity pocket. When the inner barrier is broken as shown in the inward-open conformation, the sugar selectivity pocket is also broken. The binding assays by isothermal titration calorimetry revealed that this inward-facing conformation trapped by the conformation-selective Nb exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for the substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is also supported by molecular dynamics simulations. Furthermore, the use of this Nb in combination with the hydron/deuterium exchange mass spectrometry allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

molecular biology↗

bioRxiv2020

Molecular recognition of sugar binding in a melibiose transporter MelB by X-ray crystallography

The symporter melibiose permease MelB is the best-studied representative from MFS_2 family and the only protein in this large family with crystal structure determined. Previous thermodynamic studies show that MelB utilizes a cooperative binding as the core mechanism for its obligatory symport. Here we present two sugar-bound X-ray crystal structures of a Salmonella typhimurium MelB D59C uniport mutant that binds and catalyzes melibiose transport uncoupled to either cation, as determined by biochemical and biophysical characterizations. The two structures with bound nitrophenyl--D-galactoside or dodecyl-{beta}-D-melibioside, which were refined to a resolution of 3.05 or 3.15 [A], respectively, are virtually identical at an outward-facing conformation; each one contains a -galactoside molecule in the middle of protein. In the substrate-binding site, the galactosyl moiety on both ligands are at an essentially same configuration, so a galactoside specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for the binding of sugar in MelB is deciphered. The data also allow to assign the conserved cation-binding pocket, which is directly connected to the sugar specificity determinant pocket. The intimate connection between the two selection sites lays the structural basis for the cooperative binding and coupled transport. This key structural finding answered the long-standing question on the substrate binding for the Na+-coupled MFS family of transporters. SignificanceMajor facilitator superfamily_2 transporters contain >10,000 members that are widely expressed from bacteria to mammalian cells, and catalyze uptake of varied nutrients from sugars to phospholipids. While several crystal structures with bound sugar for other MFS permeases have been determined, they are either uniporters or symporters coupled solely to H+. MelB catalyzes melibiose symport with either Na+, Li+, or H+, a prototype for Na+-coupled MFS transporters, but its sugar recognition has been a long-unsolved puzzle. Two high-resolution crystal structures presented here clearly reveal the molecular recognition mechanism for the binding of sugar in MelB. The substrate-binding site is characterized with a small specificity groove adjoining a large nonspecific cavity, which could offer a potential for future exploration of active transporters for drug delivery.

biophysics↗